Yayoi Idaira et al.: Zinc Transporter ZIP13 in Degenerative Changes in PDL and Alveolar Bone Journal of Hard Tissue Biology 25[1] (2016) 49- 56 © 2016 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original

Role of Zinc Transporter ZIP13 in Degenerative Changes in Periodontal Ligament and Alveolar Bone

Yayoi Idaira1), Takaaki Munemasa1), Toshiyuki Fukada2,3,4), Shinji Shimoda5) and Yoshinobu Asada1)

1) Department of Pediatric , Tsurumi University, School of Dental Medicine, Yokohama, Japan 2) Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan 3) Department of Oral Pathology and Diagnostics, Showa University, School of Dentistry, Tokyo, Japan 4) RIKEN Center for Integrative Medical Sciences, Yokohama, Japan 5) Department of Oral Anatomy, Tsurumi University, School of Dental Medicine, Yokohama, Japan (Accepted for publication, October 13, 2015)

Abstract: ZIP13, a zinc transporter encoded by Slc39a13, acting on the homeostasis of zinc implicated in the biosynthesis and modification of collagen secretion, and the morphogenesis of connective tissues. This study aimed to determine the morphological changes in periodontal ligament and alveolar bone subjected to mechanical stress such as mastication, considering the functional role of ZIP13 in age-controlled periodontal tissue. of wild type (WT) and knock-out (KO) mice were excised 2,4,6,12,20 and 50 weeks the periodontal ligament and alveolar bone surrounding the root of the first molar were subjected to light microscopy, transmission electron microscopy and immunohistochemistry. We found that the of KO mice exhibited several morphological changes such as connective tissues degenerative changes in during the early stage, suggesting that the lack of ZIP13 caused degenerative changes in homeostasis of collagen during the early stage, which could worsen upon application of mechanical stress. Consequently, ZIP13 was considered as one of the genetic factors in the control of the development and maturation of periodontal tissues.

Key words: ZIP13, Zip13-KO mice, Periodontal ligament, HE, Collagen fiber

Introduction human6-8). Morphogenetic abnormalities in KO mice include Zinc is one of the essential trace elements in regulating the growth retardation, kyphosis, weakened skin, decrease in bone maturation of biological tissues. Deficiency during growth phase mass, abnormal oral cavity, dysplastic incisors, occlusal has been known to cause severe growth failure1), such as delayed abnormalities, fractured teeth, abnormal root dentin and irregular wound healing and dysgeusia. Furthermore, zinc- deficiency is shape of molar crowns. Moreover, ZIP13 was detected known to be related to aging process particularly that of collagen intracellularly in golgi apparatus of osteoblasts, odontoblasts and fibers2, 3). Zinc transporters act in the homeostasis of zinc both fibroblasts. Hence, ZIP13 is thought to be involved in the intracellularly and extracellularly. Slc39/Zip family zinc modification and biosynthesis of secreted collagen and is transporters increase the zinc level in cytosol, on the other hand, concluded to be the gene that affects the morphogenesis of Slc30/ZnT family zinc transporters reduce zinc levels in the biological connective tissues6). cytoplasm4). Recently, several functional analysis of zinc In this study, we focused on the abnormalities of collagen in transporter genes in mutant mice were carried out, some of which KO mice specifically, the temporal changes in the morphology of actually led to the gradual clarification of the mechanism in the the periodontal ligament and alveolar bone, as well as the control of homeostasis in the growth of mammalian individuals5). functional role of Zip13 gene in the aging process of periodontal Slc39a/Zip13 knockout (KO) mice which were prepared according tissues subjected to mechanical stress such as mastication. to the study by Fukada et al. is a typical example to elucidate the Furthermore, morphological changes in the connective tissues in function of zinc transporter in vitro and in vivo, from mouse to triceps (Achilles tendon) were also investigated in response to mechanical stress. Correspondence to: Dr Yayoi Idaira, Department of pediatric dentistry, Tsurumi University School of dental medicine, 2-1-3 Tsurumi, Tsurumi- ku Yokohama, 230-8501 Japan; Tel: +81-45-580-8401; Fax: +81-45-573- Materials and Methods 9599; E-mail: [email protected] This experiment was performed in accordance with Tsurumi 49 J.Hard Tissue Biology Vol. 25(1):49 -56, 2016 University Laboratory Animal Guidelines. Wild type (WT) and The mandibles of WT and KO mice at 2, 4, 6 and 12 week KO mice2,3,5,6) were used in this experiment. were excised, decalcified, embedded in paraffin and sectioned. Anti-collagen 1 (Abcam, ab34710) was the primary antibody used Light microscopic analysis and normal rabbit Ig for the negative control. Briefly, sections Observation of periodontal ligament and alveolar bone by HE were treated with antigen activation enzyme, followed by primary staining antibody with a concentration of 0.04 μ/ml and the antigenic sites The mandibles of WT and KO mice at 2, 4, 6, 12, 20 and 50 of collagen fibers in periodontal ligament and alveolar bone in weeks (including the first molars) were excised (total n=24). After the first molar were observed. decalcification, the tissues were embedded in paraffin and sectioned in a buccolingual direction. Then after, sections were Results stained with hematoxylin and eosin (HE) and examined under the Temporal changes in the periodontal ligament space and light microscope. Silver impregnation was also done to examine alveolar bone further the periodontal ligament fibers, alveolar bone and General observation in light microscopy periodontal ligament cells. In HE staining, at 2 weeks, in both WT and KO mice, the first molar was still inside the and has not erupted yet. In Percentage of bone marrow cavity of alveolar bone WT mice, there was no change in the width of the periodontal An image software (Image-Pro Plus, version 6.2) was used to ligament from the cervical to the apical portion over time and was calculate the proportion of bone marrow region in HE sections in almost constant until 20 weeks. Although KO mice at 6 and 12 the following manner. weeks showed similar tendency with WT mice, at 20 and 50 weeks Percentage of bone marrow = 1-A/B significant irregularities in alveolar bone wall as well as in the A=area of the bone marrow root apex were apparent (Fig. 1-a,b). As age increased, periodontal B=area of the alveolar bone (length [from the alveolar crest to the ligament space became narrower and adhesion sites appeared. root apex] × width [the width between the two points parallel to Silver staining in the periodontal ligament near the alveolar crest the long axis of the tooth perpendicular to the surface of the in WT and KO mice at 2 weeks showed that Sharpey’s fibers alveolar bone on the buccal and lingual sides]) attached to alveolar bone specifically to the bundle bone and lamellated bone exhibited a three-dimensional porous sieve-like Transmission Electron microscopic (TEM) analysis appearance. In WT mice, the space between bundle bone and Ultrastructure of the periodontal ligament fibers periodontal ligament on the buccal side has a constant width with WT and KO mice at 6, 12 and 20 weeks old were sacrificed lots of Sharpey’s fibers. A huge difference was observed over time under anesthetic by perfusion and the tissues were fixed using (from 4 to 50 weeks). In WT mice at 6, 12, 20 and 50 weeks, the half-Karnovsky fixative solution (2.5 % glutaraldehyde, 2 % fibroblasts in the periodontal ligament exhibit spindle in shape paraformaldehyde, 0.1M cacodylate buffer). Then after, the excised with flat nucleus and are densely arranged parallel to one another running from the to alveolar bone. However, KO mice mandibles were post-fixed in OsO4 for 2 hours, dehydrated in increasing ethanol series in a routine manner, embedded in resin at 6 weeks, had irregular bundle bone, and thinner and fewer and prepared for ultrathin sections. The specimens were double Sharpey’s fibers compared to WT mice (Fig. 2). Moreover, from stained with Uran Lead and the periodontal ligament fibers at the the alveolar crest to the root apex of the buccal alveolar bone, a alveolar crest were examined under TEM (JEM-1400, JEOL, deep finger-like recession occurred in a complex manner with Tokyo, Japan). reduced alveolar bone making the entire bone mass significantly The diameter of the collagen fibers in transverse section less. At 20 weeks, the alveolar crest became significantly thin and perpendicular to the long axis of the fibers was observed under further irregular outline. At 50 weeks, there was resorption of the TEM. The bundles of collagen fiber per unit area as well as periodic crest of the alveolar bone thus, a lost in the support of the tooth. striation of collagen fibers were measured using image analysis However, extensive bone changes in the outer plate of the alveolar software. The two tailed student’s test was used to analyze bone were not detected. The fibroblasts synthesized in the difference between 2 groups. periodontal ligament at 6 and 12 weeks, were less in quantity compared to those of the WT mice, spindle in shape and arranged Periodic striation of Achilles tendon parallel to the periodontal ligament fibers. However, some The Achilles tendon of WT and KO mice at 12 weeks was fibroblasts with oval nuclei contain higher percentage of observed under TEM for periodic striation, which was measured cytoplasm. At 50 weeks, the fibroblasts became atrophied and using image software (Image-Pro Plus, version 6.2). cell arrangement was disrupted.

Immunohistochemical analysis Collagen fibers of the periodontal ligament 50 Yayoi Idaira et al.: Zinc Transporter ZIP13 in Degenerative Changes in PDL and Alveolar Bone

Figure 1. Periodontal ligament space of first molar at 50weeks (HE staining).a: WT mouse; b: KO mouse. AC: alveolar crest; PL: periodontal ligament; D: dentine. Scale bar= 300 m.

Diameter of collagen fibers (Fig. 3) Alveolar bone mass (Fig. 6) Both WT and KO mice at 12 weeks had collagen fibers with When the percentage of bone cavity was subtracted to the total large diameter although this significantly decreased at 20 weeks. alveolar bone, the alveolar bone mass of KO mice was evidently The collagen fibers of WT mice at 6 and 20 weeks were less compared to WT mice. Alveolar bone mass in both groups significantly larger compared to KO mice. However, no significant increased at 12 weeks but decreased at 20 weeks. There was an difference was observed between the two groups at 12 weeks. increase in the difference in the alveolar bone mass between the two groups. Number of collagen fibers (Fig. 4) No significant difference in the number of collagen fibers in Discussion WT mice was obtained when each age group (6, 12, 20 weeks) Developmental changes during growth phase cause modification was compared. In KO mice, a significant difference was only in the living body over time and degenerative changes occur during observed at 12 weeks but not at 6 and 20 weeks. When the two the aging process of tissues. Although periodontal tissues groups were compared, more collagen fibers seen in WT mice continually suffer from mechanical stress caused by mastication, than KO mice and significant difference were observed at 6 and in this experiment, the KO mice resulted to a number of 20 weeks but not at 12 weeks. morphological changes in the periodontium at an early stage compared to WT mice. Previous researches on Width of collagen fibers (Fig. 5) mentioned that resorption of the alveolar bone, which is one of At 12 weeks, the average width of the collagen fibers in the the signs of periodontal disease is said to be a degenerative change. periodontal ligament in WT mice was 67.7 nm ± 2.94 compared In this regard, in KO mice, 12 weeks appeared to be the turning to 63.89 nm ± 3.15 in KO mice. The difference in the width of point, which marked the beginning of morphological changes collagen fibers was statistically significant. In the Achilles tendon, advancing to periodontal disease considered as part of degenerative the average was 75.7 nm in WT mice and 64.2 nm in KO mice in changes. In the present study, we focused on the morphological which WT mice has significantly greater width compared to KO changes in the alveolar bone and root in Slc39a13/Zip13 KO mice. mice. Alveolar bone Immunolocalization of col1 In the study of Sakai et al. alveolar bone grew normally until 20 Fibroblasts in the periodontal ligament and alveolar bone in weeks and then later, degenerative changes occurred indicated both WT and KO mice were positive to col1 (figure not shown). by decrease in bone mass and irregular alveolar bone and 51 J.Hard Tissue Biology Vol. 25(1):49 -56, 2016

Figure 2. Temporal changes in periodontal ligament fibers around the alveolar crest (silver staining). a to d: WT mouse; e to h: KO mouse. a and e: 6 weeks; b and f: 12 weeks; c and g: 20 weeks; d and h: 50 weeks. Scale bar= 10 m. AC: alveolar crest ; PL: periodontal ligament; C: cement; D: dentine

52 Yayoi Idaira et al.: Zinc Transporter ZIP13 in Degenerative Changes in PDL and Alveolar Bone

65 150 WT KO WT KO

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m 6w 12w 20w a i 47 D Figure 4. Temporal changes in the number of collagen per unit 0 area (*p<0.05). 6w 12w 20w Figure 3. Temporal changes in the diameter of collagen fibrils of the periodontal ligament (*p<0.05). 700 WT KO ) 650 m 90 n ( Periodontal ligament )

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P 0 0 WT KO 6w 12w 20w Figure 5. Periodic width of collagen fibers ( periodontal ligament, Figure 6. Temporal changes in bone mass around the first molars tendon) in WT and KO mice at 12 weeks (*p<0.05). in WT and KO mice. n=3.

periodontal ligament9). We observed the morphological changes described11). This study likewise showed irregularities in the width in alveolar bone of WT mice at 20 weeks, however the degenerative of the periodontal ligament space and alveolar in KO mice. Since changes in alveolar such as coarse or irregular contour only Ishikawa et al. showed degenerative changes in periodontal occurred at 50 weeks. In KO mice, irregular alveolar bone at the ligament and alveolar bone in rats due to the lack of magnesium root apex occurred early at 12 weeks and continued to progress at and the present study showed the same changes due to lack of 50 weeks. Rapid degenerative changes in KO mice are caused by zinc, using the role of bivalent cation of zinc and magnesium in defect in bone metabolism due to absence of Zip13, which seemed cell, it was assumed that the trace elements are essential in the to be related to the inability to maintain homeostasis. extracellular matrix and their deficiency caused degenerative changes. Although there is a difference in the animal models used, Periodontal Ligament space similar findings were obtained. The periodontal ligament space is maintained constantly at a The periodontal ligament attaches the tooth to the alveolar physiological width, but due to aging, narrowing of the periodontal bone and acts as a cushion against mechanical stress applied to ligament space occurs as one of the degenerative processes in the tooth by forces of mastication. As weeks passed by in KO humans10). The major change in the periodontal ligament space in mice, Sharpey’s fibers became thinner, fewer and disorderly WT mice was observed at 20 weeks. The change in the width of arranged. It is probable that collagen fibers were more fragile the periodontal ligament in KO mice was almost constant at 12 compared to usual collagen fibers, in which it was more likely weeks and later became irregular at 20 weeks. According to the difficult to sustain pressure from normal occlusion and masticatory study of Ishikawa et al. remodeling of alveolar bone in Mg- force. Although it was not shown in the results, we continued deficient mice occurred at 8 weeks in which irregularity in the breeding after 50 weeks, it was confirmed that collagen fibers width of the periodontal ligament and alveolar bone was were extremely fragile to maintain the extracellular matrix. 53 J.Hard Tissue Biology Vol. 25(1):49 -56, 2016 Although, there was no adhesion between the alveolar bone and for their supports, and excellent technical assistances of Genostaff. cementum, the periodontal ligament space was very narrow. Conflict of Interest Periodontal ligament in alveolar crest The authors have declared that no COI exists. In humans, the collagen in the skin becomes thinner and less in number during aging12). The results of the present study showed References that the quantity and width of collagen fibers in KO mice at 12 1. Prasad AS. Discoverry of human zinc deficiency and studies weeks significantly decreased. With silver impregnation, the in an experimental human model. Am J Clin Nutr 53: 403- change in the periodontal ligament and alveolar bone was evident 412, 1991 from 12 weeks, and at 20 weeks, the alveolar crest wall became 2. Fukada T, Yamasaki S, Nishida K, Murakami M and Hirarno T. irregular; alveolar crest fibers and horizontal fibers became Zinc homeostasis and signaling in health and desease: Zinc obscurely less. Reduction in the number of collagen fibers as well signaling. J Biol Inorg Chem 16: 1123-1134, 2011 as loss in the width of the periodontal membrane was also slightly 3. Fukada T and Kanbe T. Molecular and genetic features of observed in WT mice at 20 weeks but there was no disturbance in zinc transporters in physiology and pathogenesis. Metallomis the periodontal ligament fibers running to the alveolar bone crest. 3: 662-674, 2011 The morphological changes in KO mice at 50 weeks were not 4. Kambe T, Yamaguchi-Iwai T, Sasaki R and Nagao M. seen in WT mice. Although morphological changes in alveolar Overview of mammalian zinc transporters. Cell Moll Life bone occurred both in WT and KO mice, morphological changes Sci 61: 49-68, 2004 occurred earlier and the progress was faster in KO mice. The 5. Fukada T, Hojyo S and Bin BH. Zinc signal in growth results suggest that knockout of Zip13 lead to the metabolic control and bone diseases. In Zinc Signals in Cellular abnormalities in collagen and the lack of homeostasis brought Functions and Disorders, ed by Fukada T and Kambe T, about rapid degenerative changes. Inc., Tokyo: Springer, 2014, pp249-267. 6. Fukada T, Civic N, Furuichi T, Shimoda S, Mishima K, Periodic width of collagen fiber Higashiyama H, Idaira Y, Asada Y, Kitamura H, Yamasaki At 12 weeks, the width of collagen fibers in the periodontal S, Hojyo S, Nakayama M, Ohhara O, Koseki H, dos Santos ligament and at the Achilles tendon in WT mice are significantly HG, Bonafe L, Ha-Vinh R, Zankl A, Unger S, Kraenzlin larger compared to KO mice. Whether time is a factor in the ME, Beckman JS, Saito I, Rivolta C, Ikegawa S, Superti- difference in the width is uncertain. According to Fukuda et al.6), Furga A and Hirano T. The Zinc transporter SLC39A13/ modification of the cross-linking of collagen is due to the defect ZIP13 is required for connective tissue development; its in Zip13 wherein the amino acid sequence of disulfide bonds and involvement in BMP/TGF signaling pathways. Plos one 3: polypeptide chains might have affected the collagen fibril e3642, 2008 formation in the extracellular matrix. 7. Bin BH, Fukada T, Hosaka T, Yamasaki S, Ohashi W, Hojyo Zip13 gene is involved in the biosynthesis and modification S, Miyai T, Nishida K, Yokoyama S and Hirano T. of secretion of collagen, essential for homeostasis in living body. Biochemical characterization of human ZIP13 protein: A The study presented the temporal changes in the periodontium homo-dimerized zinc transporter involved in the exposed to long-term mechanical stress through mastication. The spondylocheiro dysplastic Ehlers-Danlos syndrome. J Biol failure to maintain homeostasis of collagen was due to the lack Chem 286: 40255-40265, 2011 Zip13 gene. Failure of the cross-linking of collagen secreted by 8. Bin BH, Hojyo S, Hosaka T, Bhin J , Kano H, Miyai T, Ikeda the fibroblasts in the extracellular matrix lead to the aberration in M, Kimura-Someya T, Shirouzu M, Cho EG, Fukue K, the modification, resulting to thin and fewer collagen fibers and Kambe T, Ohashi W, Kim KH, Seo J, Choi DH, Nam YJ, highly vulnerable to degenerative changes. Moreover, this lead Hwang D, Fukunaka A, Fujitani Y, Yokoyama S, Superti- to more rapidly degenerative changes in the connective tissues at Furga A, Ikegawa S, Lee TR and Fukada T. Molecular an early stage when the periodontal ligament is subjected to pathogenesis of spondylocheirodysplastic Ehlers-Danlos mechanical stress. Therefore, ZIP13 is considered to be important syndrome caused by mutant ZIP13 proteins. EMBO in the control of aging of periodontal tissue. MoleculMedi 6: 1028-1042, 2014 9. Sakai T, Kido M, Terada Y, Kawano K, Ohsaki Y and Kurisu Acknowledgment K. Age dependent changes in the periodontal ligament of The experiment is a grant in aid scientific research (Project mice -Morphological and immunofluorescent studies-. J Jpn Number: 22592302). The authors thank Ms. Toshie Chiba, Mr. Prothdont Soc 35: 794-803, 1991 Masaru Emura and Mr. Junichi Yamazoe (Tsurumi University) 10. Van der Velden. Effect of age on the periodontium. J C lin

54 Yayoi Idaira et al.: Zinc Transporter ZIP13 in Degenerative Changes in PDL and Alveolar Bone Periodont 11: 281-294, 1984 12. Castel-Branco C, Pons F, Gratacos E, Fortuny A, Vanrell JA 11. Ishikawa M, Shimoda S and Nakamura Y. Histological and Gonsales-Mendo J. Relationship between skin collagen study of the periodontal ligament and alveolar bone in and bone change during aging. Maturitas 18: 199-206, 1994 magnesium-dificient rats. J Oral biosci 52: 170-180, 2010

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