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epithelial rest of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth
Accepted Manuscript Ameloblast-like characteristics of human Hertwig's epithelial rest of Malassez/ epithelial rest of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth Hyun Nam, Gee-Hye Kim, Jae-Won Kim, Jae Cheoun Lee, Kyunghoon Lee, Sun-Ho Lee PII:
S0006-291X(17)31209-3
DOI:
10.1016/j.bbrc.2017.06.077
Reference:
YBBRC 37983
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 June 2017 Accepted Date: 14 June 2017
Please cite this article as: H. Nam, G.-H. Kim, J.-W. Kim, J.C. Lee, K. Lee, S.-H. Lee, Ameloblastlike characteristics of human Hertwig's epithelial rest of Malassez/epithelial rest of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.077. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE Ameloblast-like characteristics of human Hertwig’s epithelial rest of Malassez/epithelial rest
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of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth
AUTHORS
Hyun Nam1,2,3, Gee-Hye Kim4, Jae-Won Kim4, Jae Cheoun Lee5, Kyunghoon Lee3,6,*, and
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Sun-Ho Lee1,2,*
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AFFILIATIONS
Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University, Seoul
06351, South Korea 2Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, South Korea 3Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, 16419, South
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Korea 4Laboratory of Molecular Genetics, Dental Research Institute, School of Dentistry, Seoul National University, Seoul 03080, Korea 5Children’s Dental Center and CDC Baby
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Tooth Stem Cell Bank, Seoul 06072, South Korea, South Korea 6Department of Anatomy &
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Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea
* CORRESPONDENCE Sun-Ho Lee, MD, PhD
Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 06351, South Korea Tel: (82-2) 3410-2457; Fax: (82-2) 3410-0048; E-mail addresses: [email protected]
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ACCEPTED MANUSCRIPT Kyunghoon Lee, MD, PhD Department of Anatomy & Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea
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Tel: (82-31) 299-6070; Fax: (82-31) 299-6089; E-mail addresses: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
Tooth develops through reciprocal interactions between oral epithelium and oral
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mesenchyme. To regenerate tooth, both components are necessary to mimic epithelialmesenchymal interactions. In adult teeth, Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells which are derived from oral
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epithelium. In this study, we investigated whether HERS/ERM cells could have ameloblastlike characteristics via interaction with stem cells from human exfoliated deciduous teeth
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(SHEDs) as an oral mesenchymal component. HERS/ERM cells were co-cultured with SHEDs in three different ratios (1:1, 2:1, 3:1) in non-osteogenic or osteogenic culture condition. During co-culturing, the expression of amelogenin and E-cadherin was colocalized in HERS/ERM cells, suggestive of ameloblast-like characteristics of HERS/ERM
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cells, because HERS/ERM cells alone or SHEDs alone did not express amelogenin. Moreover, at 21 days after co-culturing, calcium deposits were observed in both HERS/ERM cells and SHEDs, and they increased proportional to the number of HERS/ERM cells. When
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HERS/ERM cells and SHEDs were co-transplanted in vivo at three different ratios (1:1, 2:1, 3:1), the expression of amelogenin was observed and it increased proportional to the number
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of HERS/ERM cells. Taken together, our results suggested that HERS/ERM cells might have ameloblast-like characteristics via interaction with oral mesenchymal component such as SHEDs.
KEYWORDS HERS/ERM cell; SHEDs; Ameloblast; Epithelial-mesenchymal interaction; Amelogenin
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ACCEPTED MANUSCRIPT 1. INTRODUCTION
Tooth develops through reciprocal interactions between oral epithelium and oral
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mesenchyme, which were also observed in the developmental process of other organs such as feather, hair follicles, and mammary glands [1, 2]. Epithelial-mesenchymal interactions regulate initiation, differentiation, and morphogenesis of teeth during development, and
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growth factors, receptors, and signaling pathways have been well studied, which are involved in epithelial-mesenchymal interactions have been well studies [3]. For the therapeutic
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regeneration of teeth, epithelia-mesenchymal interaction is necessary like developmental process [4]. For oral mesenchymal component, stem cells from human exfoliated deciduous teeth (SHEDs) and dental pulp stem cells (DPSCs) may be feasible sources, because both of them have mesenchymal stem cell (MSC)-like characteristics and make dentin-like structures
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in vivo [5, 6]. On the contrary, there are limited sources as oral epithelial components, because enamel-forming cells have not been identified from adult teeth.
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ERM is located in the periodontal ligament tissue near the root and are derived from the Hertwig's epithelial root sheath (HERS) fragments during root development. HERS/ERM
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cells are unique epithelial cells in adult teeth and they are maintained in the periodontium of adult teeth [7, 8]. The functional roles of HERS/ERM cells are involved in the homeostasis of periodontium and especially in the regeneration of cementogenesis [9-11]. HERS cells regulate the differentiation and proliferation of periodontal ligament stem cells and even could form cementum through epithelial-mesenchymal transition (EMT) [12, 13]. Recently, the stem cell-like characteristics of HERS/ERM cells were reported and especially, they may contain epithelial stem cell population [14-16]. Considering the developmental origin of 4
ACCEPTED MANUSCRIPT HERS/ERM cells, it is postulated that HERS/ERM cells might have ameloblast-like characteristics. In this study, we investigated whether human HERS/ERM cells might have ameloblast-like characteristics and like development of teeth, epithelial-mesenchymal
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interactions with SHEDs as an oral mesenchymal component were necessary to acquire
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ameloblast-like characteristics.
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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS
2.1. Primary isolation and culture of HERS/ERM cells and SHEDs
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The experimental protocol was approved by the Institutional Review Board (S-D20070004). Informed consent was obtained from the patients. Human third molars and deciduous teeth were delivered in Hank’s balanced salt solution (HBSS) (Welgene, Daegu, Korea)
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supplemented with 3% Antibiotics antiomycotics (Gibco, Grand Island, NY, USA) at 4°C. HERS/ERM cells and SHEDs were isolated and cultured as previous reported [5, 16-18].
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Briefly, periodontal ligament tissues and dental pulp were extracted with fine forcepts, followed by mechanical mincing and enzymatic digestion with 1 mg/mL of Collagenase type I (Grand Island, NY, USA) and 2.4 mg/mL of Dispase (Grand Island, NY, USA) at 37°C for 1 hour. After inactivation of enzyme, cells were washed two times. Single-cell suspensions
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were maintained in serum-free keratinocyte growth medium (KGM; Lonza Rockland, Rock Island, ME) and minimum essential medium alpha (α-MEM; Hyclone, Road Logan, Utah, USA) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone) to acquire HERS/ERM
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70% confluency.
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cells and SHEDs, respectively. Medium was changed every 2 days. Cells were sub-cultured at
2.2. Immunofluorescent staining Cells were fixed with ice-cold methanol for 10 minutes at -20°C. Slides were washed with PBS, blocked with 10% normal goat serum for 1 hour at room temperature. We used rabbitanti-E-cadherin (1:100; Santa Cruz Biotechnology Inc., CA, USA), rabbit-anti-N-cadherin (1:100; Santa Cruz Biotechnology Inc.), mouse anti-amelogenin (1:100; Santa Cruz Biotechnology Inc.), Alexa 488-conjugated goat-anti-rabbit IgG (1:700; Molecular Probes, 6
ACCEPTED MANUSCRIPT Eugene, OR, USA), and Alexa 594-conjugated goat-anti mouse IgG (1:700; Molecular Probes) antibody. Primary antibodies were applied for overnight at 4°C. After washing with PBS three times, secondary antibodies were applied for 1 hour at room temperature and slides
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were washed with PBS three times. DAPI (Sigma–Aldrich, St. Louis, MO, USA) was used to stain nucleus and mounted with Flourescent Mounting Medium (Dako, Carpinteria, CA, USA). Slides were observed using a fluorescent microscope (Fluoview FV 300, Olympus,
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Japan).
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2.3. Co-culture of primary HERS/ERM cells and SHEDs
HERS/ERM cells and SHEDs at passage 3 were seeded at three different ratios (1:1. 2:1. 3:1). After co-culturing in α-MEM and 10% FBS for 2-3 days to be sub-confluent, mixed cells were maintained in non-osteogenic medium containing α-MEM and 5% FBS, or
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osteogenic medium containing α-MEM and 5% FBS supplemented with 10 mM βglycerophosphate, 50 µg/mL L-ascorbic acid phosphate), and 0.1 µM dexamethasone (all from Sigma–Aldrich, St. Louis, MO, USA) for 21 days. Medium was changed every 2 day.
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To identify calcium deposits, cells were fixed with 4% formalin and stained with 2% Alizarin
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Red (Sigma-Aldrich).
2.4. In vivo transplantation All experiments using animals followed protocols approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-1010046). Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. Previously, HERS/ERM cell line was generated by immortalization by SV40, which was used for in vivo transplantation [15]. HERS/ERM cells 7
ACCEPTED MANUSCRIPT and SHEDs were mixed at three different ratios (1:1, 2:1, 3:1) to be total 5.0 × 106 cells and transplanted with 40 mg of hydroxyapatite/Tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc., Warsaw, IN, USA) subcutaneously into 8-week-old immunocompromised mice
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(NIH-bg-nu/nu-xid. Harlan Sprague Dawley, Indianapolis, IN, USA). After 6 and 12 weeks, grafts were removed, fixed with 4% paraformaldehyde, and decalcified with buffered 10% EDTA. After embedding in paraffin, paraffin block was sectioned on a microtome (Leica,
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Wetzlar, Germany) at 5-µm thickness.
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2.5. Immunohistochemistry
Slides were deparaffinized in histoclear (National Diagnostics, Somerville, New Jersey) and rehydrated through a series of graded alcohols and distilled water. Slides were incubated in 3% hydrogen peroxide in methanol for 30 minutes to quench endogenous peroxidase. After
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blocking with 10% normal goat serum for 1 hour, primary antibody, mouse anti-amelogenin (1:100; Santa Cruz Biotechnology Inc.) was treated at 4°C overnight. After washing three times, slides were exposed to biotinylated secondary antibody for 1 hour and then avidin-
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biotin complex (Vector Laboratories) for 1 hour. Slides were stained with DAB (3,3’diaminobenzidine tetrahydrochloride) and then were counterstained with hematoxylin, and
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were scanned using the Scanscope AT (Aperio Technologies Inc., Visa, CA).
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ACCEPTED MANUSCRIPT 3. RESULTS
3.1. Primary isolation and characterization of HERS/ERM cells and SHEDs
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HERS/ERM cells and SHEDs were primarily isolated and cultured according to previous reports [16, 18]. Primarily isolated HERS/ERM cells and SHEDs showed typical epithelial cell-like and fibroblast-like morphology, respectively (Fig 1A). HERS/ERM cells showed
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growth regression at late passage (Fig 1B). However, SHEDs showed linear growth curve during culture period (Fig 1B). To characterize HERS/ERM cells and SHEDs, the expression
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of E-cadherin, N-cadherin, Snail, and Amelogenin was determined by immunofluorescent staining. As shown in Fig. 1C, HERS/ERM cells expressed E-cadherin but not N-cadherin. SHEDs expressed N-cadherin but not E-cadherin. Both cell types did not express
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Amelogenin.
3.2. Optimization of co-culturing conditions of HERS/ERM cells and SHEDs To mimic epithelial-mesenchymal interaction during development of tooth, HERS/ERM
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cells and SHEDs were co-cultured. To decide optimal ratio of co-culturing, HERS/ERM cells and SHEDs were seeded at three different ratios (1:1. 2:1, 3:1). At next day of initial seeding,
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HERS/ERM cells colonized spontaneously among SHEDs (data not shown). After 2-3 days to be confluent, the culture medium was replaced with non-osteogenic or osteogenic medium (Fig. 2A and 2B). During co-culturing period, the size of HERS/ERM cells increased in both medium, suggestive of proliferation of HERS/ERM cells. However, HERS/ERM cells cultured in osteogenic medium showed clearer margin and smaller cellular size than nonosteogenic medium, which implied better proliferative potential of osteogenic culture condition. These data suggested that although HERS/ERM cell could not proliferate in 9
ACCEPTED MANUSCRIPT serum-containing medium, HERS/ERM cells proliferated via co-culturing with SHEDs. We decided optimal co-culturing ratio of HERS/ERM cells and SHEDs as 2:1, because 3:1 or 1:1
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ratio led to the overgrowth of SHEDs or HERS/ERM cells, respectively.
3.3. In vitro ameloblast-like characteristics of HERS/ERM cells by co-culturing with SHEDs
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To determine ameloblast-like characteristics of HERS/ERM cells, the expression of amelogenin was analyzed during co-culturing period. As shown in Fig 1C, HERS/ERM cells
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and SHEDs did not express amelogenin. The expression of Amelogenin was determined at day 7 and 14 after co-culturing of HERS/ERM cells and SHEDs in non-osteogenic or osteogenic medium. As shown in Fig. 3A and 3B, the expression of Amelogenin was colocalized with E-cadherin, which suggested that during co-culturing period, the expression of
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Amelogenin and E-cadherin was maintained and co-localized within the colonies of HERS/ERM cells. At day 21 after co-culturing of HERS/ERM cells and SHEDs, calcium deposits were stained with Alizarin Red. We could observe calcium deposits in both
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HERS/ERM cells and SHEDs (Fig. 4A). The number and size of calcium deposits increased proportional to the ratio of HERS/ERM cells. On the border line between HERS/ERM cells
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and SHEDs, more calcium deposits were observed, which implied that reciprocal interactions might be important to induce the expression of amelogenin in vitro. These results indicated that HERS/ERM cells could acquire ameloblast-like characteristics via co-culturing with SHEDs in vitro.
3.4. In vivo ameloblast-like characteristics of HERS/ERM cells by co-culturing with SHEDs 10
ACCEPTED MANUSCRIPT For in vivo ameloblast-like characteristics of HERS/ERM cells, HERS/ERM cells and SHEDs were co-transplanted into the dorsal region of immunodefieicnt mouse. It was difficult to acquire enough number of primarily isolated HERS/ERM cells for transplantation,
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HERS/ERM cell line, immortalized by SV40 was used [15]. HERS/ERM cell line and SHEDs were mixed at three different ratios (1:1, 2:1, 3:1) and co-transplanted into the dorsal region of immunodeficient mice. To determine ameloblast-like characteristics of HERS/ERM
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cells, the expression of Amelogenin was analyzed at 6 and 12 week after transplantation. The expression of Amelogenin increased proportional to the number of HERS/ERM cell line at 6
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and 12 week after transplantation (Fig. 4B). These data suggested that the reciprocal interaction between HERS/ERM cell line and SHEDs might be important to induce the
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expression of Amelogenin in vivo.
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ACCEPTED MANUSCRIPT 4. DISCUSSION
Epithelial-mesenchymal interaction is involved in the process of not only embryonic
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development but also regeneration process [19]. Tooth is a representative organ, which develops through epithelial-mesenchymal interactions between dental epithelium and dental mesenchyme [3]. As considering the developmental origin of human HERS/ERM cells, it
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may be possible to hypothesize that human HERS/ERM cells might contain or have differentiation potential into ameloblast, enamel-forming cells [20, 21]. To prove this
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hypothesis, we investigated ameloblast-like differentiation of human HERS/ERM cells as a dental epithelial component by co-culturing with SHEDs as a dental mesenchymal component.
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During tooth development, dental epithelium differentiates into ameloblasts through several steps known as the presecretory, secretory, and maturation stages [22]. After crown formation of the tooth is completed, the inner and outer enamel epithelial cells form a bi-
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layered epithelial sheath, called HERS. It has been suggested that HERS cells have an important role in root development [23]. In adults, HERS cells are eventually remained as
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ERM, and some of them disappear by apoptosis [24, 25]. These HERS/ERM cells are a unique population of epithelial cells in the periodontal ligament and are believed to have a crucial role in cementum repair [26]. For the specification of ameloblast-lineage, dental epithelium should interact with dental mesenchyme, which will differentiate into odontoblast. Recently, for the regeneration of teeth, co-transplantation of developing dental epithelium and mesenchyme into kidney capsule could lead to the regeneration of whole teeth [27]. To follow the developmental process of teeth, HERS/ERM cells were co-cultured or co12
ACCEPTED MANUSCRIPT transplanted with SHEDs at 1:1, 2:1, and 3:1 ratio. In previous report, direct co-culture between HERS cells and dental mesenchyme could induce ameloblast-like differentiation [20]. Because the culture medium of HERS/ERM cells SHEDs are different [15, 16], it was
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important to find optimal culture condition to maintain both cell types. Although the size of HERS/ERM cells increased during co-culturing period, they overgrew at 3:1 ratio. On the contrary, in the case of 1:1 ratio, HERS/ERM cells did not proliferate well. The optimal co-
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culturing ratio was 2:1. Because serum-containing growth medium resulted in the cell death of HERS/ERM cells (data not shown), direct contact and various secretory factors derived
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from SHEDs might have beneficial effects on the proliferation and maintenance of HERS/ERM cells during co-culturing period.
After 21 days of co-culture, calcium deposits were observed in both cells and the amount
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of calcium deposits was concentrated at interacting region between HERS/ERM cells and SHEDs. These data also suggested that reciprocal interactions between HERS/ERM cells and SHEDs might be important for the differentiation and calcification. Recently, it was reported
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that mouse HERS/ERM (mHERS/ERM) cells can form mineralized nodules and express enamel- and cementum-related genes [28]. In that report, mHERS/ERM cells were cultured
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on a feeder layer for the maintenance, proliferation, and differentiation. More recently, it was reported that sub-cultured porcine HERS/ERM could differentiate into ameloblast-like cells and generate enamel-like structures in combination with developmental dental mesenchyme [20]. Taken together, these reports suggested the importance of epithelial-mesenchymal interactions for differentiation potentials into ameloblast or odontoblast.
Amelogenin is one of the most important molecules of ameloblast. The basal expression 13
ACCEPTED MANUSCRIPT level of amelogenin in HERS/ERM cells and SHEDs was undetectable; however, the presence or absence of amelogenin is still controversial in HERS/ERM cells. Some articles described the presence of amelogenin [29-31], whereas other studies were not able to detect it
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in HERS/ERM [32-34]. These differences may be explained by cell sources, culture conditions, and detection methods. In our result of co-culturing, the expression of amelogenin increased and localized in the region of HERS/ERM cells not SHEDs. In our results of co-
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of ameloblast-like characteristics of HERS/ERM cells.
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culturing and co-transplantation, the expression of amelogenin was well observed, suggestive
In adult teeth, dental enamel cannot be repaired because there are no cells that synthesize the enamel matrix. Tissue engineering of the enamel organ would provide an important therapeutic option for recovery of enamel loss. In general, tissue engineering involves the
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expansion of specific cell populations in vitro, culturing these cells on a scaffold, and subsequent transplantation of this scaffold into the host. For this purpose, a primary culture of stem cells and ex vivo expansion enough for transplantation are the key prerequisites [35, 36].
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The successful tissue engineering of complex tooth structures will require the characterization of the specific progenitor cell populations that mediate tooth development.
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The potential of ameloblast-like characteristics of HERS/ERM cells are expected to facilitate in vitro enamel regeneration.
ACKNOWLEDGEMENT This research was supported by a grant from National Research Foundation (NRF2013R1A1A1004602 and NRF-2016R1A5A2945889), and Samsung Biomedical Research Institute grant (SMX1161411). 14
ACCEPTED MANUSCRIPT REFERENCE
[1] I. Thesleff, A. Vaahtokari, A.M. Partanen, Regulation of organogenesis. Common
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molecular mechanisms regulating the development of teeth and other organs, Int J Dev Biol, 39 (1995) 35-50.
[2] E. Fuchs, Scratching the surface of skin development, Nature, 445 (2007) 834-842.
SC
[3] I. Thesleff, Epithelial-mesenchymal signalling regulating tooth morphogenesis, Journal of cell science, 116 (2003) 1647-1648.
M AN U
[4] M.J. Honda, S. Tsuchiya, Y. Sumita, H. Sagara, M. Ueda, The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration, Biomaterials, 28 (2007) 680-689.
[5] M. Miura, S. Gronthos, M. Zhao, B. Lu, L.W. Fisher, P.G. Robey, S. Shi, SHED: stem
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cells from human exfoliated deciduous teeth, Proceedings of the National Academy of Sciences of the United States of America, 100 (2003) 5807-5812. [6] S. Gronthos, M. Mankani, J. Brahim, P.G. Robey, S. Shi, Postnatal human dental pulp
EP
stem cells (DPSCs) in vitro and in vivo, Proceedings of the National Academy of Sciences of the United States of America, 97 (2000) 13625-13630.
AC C
[7] W. Beertsen, C.A. McCulloch, J. Sodek, The periodontal ligament: a unique, multifunctional connective tissue, Periodontology 2000, 13 (1997) 20-40. [8] H.F. Thomas, Root formation, The International journal of developmental biology, 39 (1995) 231-237.
[9] B.L. Foster, T.E. Popowics, H.K. Fong, M.J. Somerman, Advances in defining regulators of cementum development and periodontal regeneration, Current topics in developmental biology, 78 (2007) 47-126. 15
ACCEPTED MANUSCRIPT [10] J. Xiong, S. Gronthos, P.M. Bartold, Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues, Periodontology 2000, 63 (2013) 217-233.
periodontal ligament, J Endod, 39 (2013) 582-587.
RI PT
[11] D. Keinan, R.E. Cohen, The significance of epithelial rests of Malassez in the
[12] W. Sonoyama, B.M. Seo, T. Yamaza, S. Shi, Human Hertwig's epithelial root sheath cells
SC
play crucial roles in cementum formation, Journal of dental research, 86 (2007) 594-599. [13] J. Xiong, K. Mrozik, S. Gronthos, P.M. Bartold, Epithelial Cell Rests of Malassez
M AN U
Contain Unique Stem Cell Populations Capable of Undergoing Epithelial-Mesenchymal Transition, Stem cells and development, (2012).
[14] T. Tsunematsu, N. Fujiwara, M. Yoshida, Y. Takayama, S. Kujiraoka, G. Qi, M. Kitagawa, T. Kondo, A. Yamada, R. Arakaki, M. Miyauchi, I. Ogawa, Y. Abiko, H. Nikawa, S.
TE D
Murakami, T. Takata, N. Ishimaru, Y. Kudo, Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties, Lab Invest, 96 (2016) 1063-1075. [15] H. Nam, J.H. Kim, J.W. Kim, B.M. Seo, J.C. Park, J.W. Kim, G. Lee, Establishment of
EP
Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium, Molecules and cells, 37 (2014) 562-567.
AC C
[16] H. Nam, J. Kim, J. Park, J.C. Park, J.W. Kim, B.M. Seo, J.C. Lee, G. Lee, Expression profile of the stem cell markers in human Hertwig's epithelial root sheath/Epithelial rests of Malassez cells, Molecules and cells, 31 (2011) 355-360. [17] J.Y. Lee, H. Nam, Y.J. Park, S.J. Lee, C.P. Chung, S.B. Han, G. Lee, The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells, In vitro cellular & developmental biology. Animal, 47 (2011) 157-164. 16
ACCEPTED MANUSCRIPT [18] H. Nam, G. Lee, Identification of novel epithelial stem cell-like cells in human deciduous dental pulp, Biochemical and biophysical research communications, 386 (2009) 135-139.
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[19] J. Liu, J.J. Mao, L. Chen, Epithelial-mesenchymal interactions as a working concept for oral mucosa regeneration, Tissue Eng Part B Rev, 17 (2011) 25-31.
[20] Y. Shinmura, S. Tsuchiya, K. Hata, M.J. Honda, Quiescent epithelial cell rests of
SC
Malassez can differentiate into ameloblast-like cells, Journal of cellular physiology, 217 (2008) 728-738.
M AN U
[21] M. Athanassiou-Papaefthymiou, P. Papagerakis, S. Papagerakis, Isolation and Characterization of Human Adult Epithelial Stem Cells from the Periodontal Ligament, Journal of dental research, 94 (2015) 1591-1600.
[22] A.G. Fincham, J. Moradian-Oldak, J.P. Simmer, The structural biology of the developing
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dental enamel matrix, J Struct Biol, 126 (1999) 270-299.
[23] J.C. Rincon, W.G. Young, P.M. Bartold, The epithelial cell rests of Malassez--a role in periodontal regeneration?, J Periodontal Res, 41 (2006) 245-252.
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[24] F.M. Wentz, J.P. Weinmann, I. Schour, The prevalence, distribution, and morphologic changes of the epithelial remnants in the molar region of the rat, J Dent Res, 29 (1950) 637-
AC C
646.
[25] H. Kaneko, S. Hashimoto, Y. Enokiya, H. Ogiuchi, M. Shimono, Cell proliferation and death of Hertwig's epithelial root sheath in the rat, Cell Tissue Res, 298 (1999) 95-103. [26] J.D. Spouge, A new look at the rests of Malassez. A review of their embryological origin, anatomy, and possible role in periodontal health and disease, Journal of periodontology, 51 (1980) 437-444. [27] E. Ikeda, R. Morita, K. Nakao, K. Ishida, T. Nakamura, T. Takano-Yamamoto, M. 17
ACCEPTED MANUSCRIPT Ogawa, M. Mizuno, S. Kasugai, T. Tsuji, Fully functional bioengineered tooth replacement as an organ replacement therapy, Proceedings of the National Academy of Sciences of the United States of America, 106 (2009) 13475-13480.
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[28] M. Zeichner-David, K. Oishi, Z. Su, V. Zakartchenko, L.S. Chen, H. Arzate, P. Bringas, Jr., Role of Hertwig's epithelial root sheath cells in tooth root development, Developmental dynamics : an official publication of the American Association of Anatomists, 228 (2003)
SC
651-663.
[29] T.G. Diekwisch, The developmental biology of cementum, Int J Dev Biol, 45 (2001)
M AN U
695-706.
[30] M. Shimonishi, J. Hatakeyama, Y. Sasano, N. Takahashi, M. Komatsu, M. Kikuchi, Mutual induction of noncollagenous bone proteins at the interface between epithelial cells and fibroblasts from human periodontal ligament, J Periodontal Res, 43 (2008) 64-75.
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[31] M. Shimonishi, J. Hatakeyama, Y. Sasano, N. Takahashi, T. Uchida, M. Kikuchi, M. Komatsu, In vitro differentiation of epithelial cells cultured from human periodontal ligament, Journal of periodontal research, 42 (2007) 456-465.
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[32] W. Luo, H.C. Slavkin, M.L. Snead, Cells from Hertwig's epithelial root sheath do not transcribe amelogenin, J Periodontal Res, 26 (1991) 42-47.
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[33] N. Mizuno, H. Shiba, Y. Mouri, W. Xu, S. Kudoh, H. Kawaguchi, H. Kurihara, Characterization of epithelial cells derived from periodontal ligament by gene expression patterns of bone-related and enamel proteins, Cell Biol Int, 29 (2005) 111-117. [34] C.D. Fong, I. Slaby, L. Hammarstrom, Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath, J Bone Miner Res, 11 (1996) 892-898. [35] C.S. Young, S. Terada, J.P. Vacanti, M. Honda, J.D. Bartlett, P.C. Yelick, Tissue engineering of complex tooth structures on biodegradable polymer scaffolds, J Dent Res, 81 18
ACCEPTED MANUSCRIPT (2002) 695-700. [36] B. Hu, A. Nadiri, S. Kuchler-Bopp, F. Perrin-Schmitt, H. Peters, H. Lesot, Tissue
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EP
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M AN U
SC
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engineering of tooth crown, root, and periodontium, Tissue Eng, 12 (2006) 2069-2075.
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Figure 1. Primary isolation and characterization of HERS/ERM cells and SHEDs.
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Primarily isolated HERS/ERM cells at passage 3 and SHEDs at passage 6 were characterized. (A) HERS/ERM cells and SHEDs showed epithelial cell-like morphology and fibroblast-like morphology, respectively. (B) The growth curves of HERS/ERM cells and
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SHEDs were compared. (C) The expression of E-cadherin, N-cadherin, and Amelogenin was
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determined by immunofluorescent staining. Magnification was × 200.
Figure 2. Optimization of co-culturing conditions of HERS/ERM cells and SHEDs To find optimal ratio of HERS/ERM cells for co-culture, HERS/ERM cells were co-cultured with SHEDs in three different ratios of 1:1. 2:1, and 3:1 in non-osteogenic medium (A) and
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osteogenic medium (B). The size of HERS/ERM cells increased in proportion to the seeding density regardless of culture conditions. Magnification was × 100. (C) At day 21, cells were stained with Alizarin Red. Calcium deposits were observed in not only HERS/ERM cells but
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also SHEDs. As the ratio of HERS/ERM cells increased, more calcium deposits were
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observed on both cell types. Magnification was × 200.
Figure 3. The expression of E-cadherin and Amelogenin during co-culturing of HERS/ERM cells and SHEDs in vitro To determine ameloblast-like characteristics of HERS/ERM cells, the expression and localization of E-cadherin and Amelogenin was determined by immunofluorescent staining during culture period. (A) When HERS/ERM cells and SHEDs were co-cultured in nonosteogenic condition, the expression of E-cadherin and Amelogenin was co-localized in 20
ACCEPTED MANUSCRIPT HERS/ERM cells. (B) When HERS/ERM cells and SHEDs were co-culture in osteogenic condition, we could observe co-localization of E-cadherin and Amelogenin in HERS/ERM
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cells. Magnification was × 200.
Figure 4. The in vitro calcification and expression of amelogenin after in vivo cotransplantation of HERS/ERM cells and SHEDs
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(A) At day 21 after co-culturing of HERS/ERM cells and SHEDs, calcium deposits were stained with Alizarin Red. Calcium deposits were observed in not only HERS/ERM cells but
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also SHEDs. As the ratio of HERS/ERM cells increased, more calcium deposits were observed on both cell types. Magnification was × 200. (B) HERS/ERM cells and SHEDs were co-transplanted at three ratios (1:1, 2:1, 3:1) into immunodeficient mouse. At 6 and 12 week after transplantation, the expression of amelogenin was observed in the region of
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extracellular matrix and was up-regulated proportional to the number of HERS/ERM cells.
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Osteogenic medium
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ACCEPTED MANUSCRIPT HERS/ERM cells are epithelial remnant of enamel-forming cells. HERS/ERM cells have ameloblast-like characteristics via interaction with SHEDs. For tooth regeneration, both HERS/ERM cells and SHEDs will be necessary for epithelial-
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mesenchymal interaction.
S0006-291X(17)31209-3
DOI:
10.1016/j.bbrc.2017.06.077
Reference:
YBBRC 37983
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 June 2017 Accepted Date: 14 June 2017
Please cite this article as: H. Nam, G.-H. Kim, J.-W. Kim, J.C. Lee, K. Lee, S.-H. Lee, Ameloblastlike characteristics of human Hertwig's epithelial rest of Malassez/epithelial rest of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.077. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE Ameloblast-like characteristics of human Hertwig’s epithelial rest of Malassez/epithelial rest
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of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth
AUTHORS
Hyun Nam1,2,3, Gee-Hye Kim4, Jae-Won Kim4, Jae Cheoun Lee5, Kyunghoon Lee3,6,*, and
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Sun-Ho Lee1,2,*
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AFFILIATIONS
Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University, Seoul
06351, South Korea 2Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, South Korea 3Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, 16419, South
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Korea 4Laboratory of Molecular Genetics, Dental Research Institute, School of Dentistry, Seoul National University, Seoul 03080, Korea 5Children’s Dental Center and CDC Baby
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Tooth Stem Cell Bank, Seoul 06072, South Korea, South Korea 6Department of Anatomy &
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Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea
* CORRESPONDENCE Sun-Ho Lee, MD, PhD
Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 06351, South Korea Tel: (82-2) 3410-2457; Fax: (82-2) 3410-0048; E-mail addresses: [email protected]
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ACCEPTED MANUSCRIPT Kyunghoon Lee, MD, PhD Department of Anatomy & Cell Biology, Sungkyunkwan University School of Medicine, Suwon, 16419, South Korea
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Tel: (82-31) 299-6070; Fax: (82-31) 299-6089; E-mail addresses: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
Tooth develops through reciprocal interactions between oral epithelium and oral
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mesenchyme. To regenerate tooth, both components are necessary to mimic epithelialmesenchymal interactions. In adult teeth, Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells which are derived from oral
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epithelium. In this study, we investigated whether HERS/ERM cells could have ameloblastlike characteristics via interaction with stem cells from human exfoliated deciduous teeth
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(SHEDs) as an oral mesenchymal component. HERS/ERM cells were co-cultured with SHEDs in three different ratios (1:1, 2:1, 3:1) in non-osteogenic or osteogenic culture condition. During co-culturing, the expression of amelogenin and E-cadherin was colocalized in HERS/ERM cells, suggestive of ameloblast-like characteristics of HERS/ERM
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cells, because HERS/ERM cells alone or SHEDs alone did not express amelogenin. Moreover, at 21 days after co-culturing, calcium deposits were observed in both HERS/ERM cells and SHEDs, and they increased proportional to the number of HERS/ERM cells. When
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HERS/ERM cells and SHEDs were co-transplanted in vivo at three different ratios (1:1, 2:1, 3:1), the expression of amelogenin was observed and it increased proportional to the number
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of HERS/ERM cells. Taken together, our results suggested that HERS/ERM cells might have ameloblast-like characteristics via interaction with oral mesenchymal component such as SHEDs.
KEYWORDS HERS/ERM cell; SHEDs; Ameloblast; Epithelial-mesenchymal interaction; Amelogenin
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ACCEPTED MANUSCRIPT 1. INTRODUCTION
Tooth develops through reciprocal interactions between oral epithelium and oral
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mesenchyme, which were also observed in the developmental process of other organs such as feather, hair follicles, and mammary glands [1, 2]. Epithelial-mesenchymal interactions regulate initiation, differentiation, and morphogenesis of teeth during development, and
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growth factors, receptors, and signaling pathways have been well studied, which are involved in epithelial-mesenchymal interactions have been well studies [3]. For the therapeutic
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regeneration of teeth, epithelia-mesenchymal interaction is necessary like developmental process [4]. For oral mesenchymal component, stem cells from human exfoliated deciduous teeth (SHEDs) and dental pulp stem cells (DPSCs) may be feasible sources, because both of them have mesenchymal stem cell (MSC)-like characteristics and make dentin-like structures
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in vivo [5, 6]. On the contrary, there are limited sources as oral epithelial components, because enamel-forming cells have not been identified from adult teeth.
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ERM is located in the periodontal ligament tissue near the root and are derived from the Hertwig's epithelial root sheath (HERS) fragments during root development. HERS/ERM
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cells are unique epithelial cells in adult teeth and they are maintained in the periodontium of adult teeth [7, 8]. The functional roles of HERS/ERM cells are involved in the homeostasis of periodontium and especially in the regeneration of cementogenesis [9-11]. HERS cells regulate the differentiation and proliferation of periodontal ligament stem cells and even could form cementum through epithelial-mesenchymal transition (EMT) [12, 13]. Recently, the stem cell-like characteristics of HERS/ERM cells were reported and especially, they may contain epithelial stem cell population [14-16]. Considering the developmental origin of 4
ACCEPTED MANUSCRIPT HERS/ERM cells, it is postulated that HERS/ERM cells might have ameloblast-like characteristics. In this study, we investigated whether human HERS/ERM cells might have ameloblast-like characteristics and like development of teeth, epithelial-mesenchymal
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interactions with SHEDs as an oral mesenchymal component were necessary to acquire
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ameloblast-like characteristics.
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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS
2.1. Primary isolation and culture of HERS/ERM cells and SHEDs
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The experimental protocol was approved by the Institutional Review Board (S-D20070004). Informed consent was obtained from the patients. Human third molars and deciduous teeth were delivered in Hank’s balanced salt solution (HBSS) (Welgene, Daegu, Korea)
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supplemented with 3% Antibiotics antiomycotics (Gibco, Grand Island, NY, USA) at 4°C. HERS/ERM cells and SHEDs were isolated and cultured as previous reported [5, 16-18].
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Briefly, periodontal ligament tissues and dental pulp were extracted with fine forcepts, followed by mechanical mincing and enzymatic digestion with 1 mg/mL of Collagenase type I (Grand Island, NY, USA) and 2.4 mg/mL of Dispase (Grand Island, NY, USA) at 37°C for 1 hour. After inactivation of enzyme, cells were washed two times. Single-cell suspensions
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were maintained in serum-free keratinocyte growth medium (KGM; Lonza Rockland, Rock Island, ME) and minimum essential medium alpha (α-MEM; Hyclone, Road Logan, Utah, USA) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone) to acquire HERS/ERM
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70% confluency.
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cells and SHEDs, respectively. Medium was changed every 2 days. Cells were sub-cultured at
2.2. Immunofluorescent staining Cells were fixed with ice-cold methanol for 10 minutes at -20°C. Slides were washed with PBS, blocked with 10% normal goat serum for 1 hour at room temperature. We used rabbitanti-E-cadherin (1:100; Santa Cruz Biotechnology Inc., CA, USA), rabbit-anti-N-cadherin (1:100; Santa Cruz Biotechnology Inc.), mouse anti-amelogenin (1:100; Santa Cruz Biotechnology Inc.), Alexa 488-conjugated goat-anti-rabbit IgG (1:700; Molecular Probes, 6
ACCEPTED MANUSCRIPT Eugene, OR, USA), and Alexa 594-conjugated goat-anti mouse IgG (1:700; Molecular Probes) antibody. Primary antibodies were applied for overnight at 4°C. After washing with PBS three times, secondary antibodies were applied for 1 hour at room temperature and slides
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were washed with PBS three times. DAPI (Sigma–Aldrich, St. Louis, MO, USA) was used to stain nucleus and mounted with Flourescent Mounting Medium (Dako, Carpinteria, CA, USA). Slides were observed using a fluorescent microscope (Fluoview FV 300, Olympus,
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Japan).
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2.3. Co-culture of primary HERS/ERM cells and SHEDs
HERS/ERM cells and SHEDs at passage 3 were seeded at three different ratios (1:1. 2:1. 3:1). After co-culturing in α-MEM and 10% FBS for 2-3 days to be sub-confluent, mixed cells were maintained in non-osteogenic medium containing α-MEM and 5% FBS, or
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osteogenic medium containing α-MEM and 5% FBS supplemented with 10 mM βglycerophosphate, 50 µg/mL L-ascorbic acid phosphate), and 0.1 µM dexamethasone (all from Sigma–Aldrich, St. Louis, MO, USA) for 21 days. Medium was changed every 2 day.
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To identify calcium deposits, cells were fixed with 4% formalin and stained with 2% Alizarin
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Red (Sigma-Aldrich).
2.4. In vivo transplantation All experiments using animals followed protocols approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-1010046). Animal experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals. Previously, HERS/ERM cell line was generated by immortalization by SV40, which was used for in vivo transplantation [15]. HERS/ERM cells 7
ACCEPTED MANUSCRIPT and SHEDs were mixed at three different ratios (1:1, 2:1, 3:1) to be total 5.0 × 106 cells and transplanted with 40 mg of hydroxyapatite/Tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc., Warsaw, IN, USA) subcutaneously into 8-week-old immunocompromised mice
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(NIH-bg-nu/nu-xid. Harlan Sprague Dawley, Indianapolis, IN, USA). After 6 and 12 weeks, grafts were removed, fixed with 4% paraformaldehyde, and decalcified with buffered 10% EDTA. After embedding in paraffin, paraffin block was sectioned on a microtome (Leica,
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Wetzlar, Germany) at 5-µm thickness.
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2.5. Immunohistochemistry
Slides were deparaffinized in histoclear (National Diagnostics, Somerville, New Jersey) and rehydrated through a series of graded alcohols and distilled water. Slides were incubated in 3% hydrogen peroxide in methanol for 30 minutes to quench endogenous peroxidase. After
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blocking with 10% normal goat serum for 1 hour, primary antibody, mouse anti-amelogenin (1:100; Santa Cruz Biotechnology Inc.) was treated at 4°C overnight. After washing three times, slides were exposed to biotinylated secondary antibody for 1 hour and then avidin-
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biotin complex (Vector Laboratories) for 1 hour. Slides were stained with DAB (3,3’diaminobenzidine tetrahydrochloride) and then were counterstained with hematoxylin, and
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were scanned using the Scanscope AT (Aperio Technologies Inc., Visa, CA).
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ACCEPTED MANUSCRIPT 3. RESULTS
3.1. Primary isolation and characterization of HERS/ERM cells and SHEDs
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HERS/ERM cells and SHEDs were primarily isolated and cultured according to previous reports [16, 18]. Primarily isolated HERS/ERM cells and SHEDs showed typical epithelial cell-like and fibroblast-like morphology, respectively (Fig 1A). HERS/ERM cells showed
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growth regression at late passage (Fig 1B). However, SHEDs showed linear growth curve during culture period (Fig 1B). To characterize HERS/ERM cells and SHEDs, the expression
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of E-cadherin, N-cadherin, Snail, and Amelogenin was determined by immunofluorescent staining. As shown in Fig. 1C, HERS/ERM cells expressed E-cadherin but not N-cadherin. SHEDs expressed N-cadherin but not E-cadherin. Both cell types did not express
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Amelogenin.
3.2. Optimization of co-culturing conditions of HERS/ERM cells and SHEDs To mimic epithelial-mesenchymal interaction during development of tooth, HERS/ERM
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cells and SHEDs were co-cultured. To decide optimal ratio of co-culturing, HERS/ERM cells and SHEDs were seeded at three different ratios (1:1. 2:1, 3:1). At next day of initial seeding,
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HERS/ERM cells colonized spontaneously among SHEDs (data not shown). After 2-3 days to be confluent, the culture medium was replaced with non-osteogenic or osteogenic medium (Fig. 2A and 2B). During co-culturing period, the size of HERS/ERM cells increased in both medium, suggestive of proliferation of HERS/ERM cells. However, HERS/ERM cells cultured in osteogenic medium showed clearer margin and smaller cellular size than nonosteogenic medium, which implied better proliferative potential of osteogenic culture condition. These data suggested that although HERS/ERM cell could not proliferate in 9
ACCEPTED MANUSCRIPT serum-containing medium, HERS/ERM cells proliferated via co-culturing with SHEDs. We decided optimal co-culturing ratio of HERS/ERM cells and SHEDs as 2:1, because 3:1 or 1:1
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ratio led to the overgrowth of SHEDs or HERS/ERM cells, respectively.
3.3. In vitro ameloblast-like characteristics of HERS/ERM cells by co-culturing with SHEDs
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To determine ameloblast-like characteristics of HERS/ERM cells, the expression of amelogenin was analyzed during co-culturing period. As shown in Fig 1C, HERS/ERM cells
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and SHEDs did not express amelogenin. The expression of Amelogenin was determined at day 7 and 14 after co-culturing of HERS/ERM cells and SHEDs in non-osteogenic or osteogenic medium. As shown in Fig. 3A and 3B, the expression of Amelogenin was colocalized with E-cadherin, which suggested that during co-culturing period, the expression of
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Amelogenin and E-cadherin was maintained and co-localized within the colonies of HERS/ERM cells. At day 21 after co-culturing of HERS/ERM cells and SHEDs, calcium deposits were stained with Alizarin Red. We could observe calcium deposits in both
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HERS/ERM cells and SHEDs (Fig. 4A). The number and size of calcium deposits increased proportional to the ratio of HERS/ERM cells. On the border line between HERS/ERM cells
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and SHEDs, more calcium deposits were observed, which implied that reciprocal interactions might be important to induce the expression of amelogenin in vitro. These results indicated that HERS/ERM cells could acquire ameloblast-like characteristics via co-culturing with SHEDs in vitro.
3.4. In vivo ameloblast-like characteristics of HERS/ERM cells by co-culturing with SHEDs 10
ACCEPTED MANUSCRIPT For in vivo ameloblast-like characteristics of HERS/ERM cells, HERS/ERM cells and SHEDs were co-transplanted into the dorsal region of immunodefieicnt mouse. It was difficult to acquire enough number of primarily isolated HERS/ERM cells for transplantation,
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HERS/ERM cell line, immortalized by SV40 was used [15]. HERS/ERM cell line and SHEDs were mixed at three different ratios (1:1, 2:1, 3:1) and co-transplanted into the dorsal region of immunodeficient mice. To determine ameloblast-like characteristics of HERS/ERM
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cells, the expression of Amelogenin was analyzed at 6 and 12 week after transplantation. The expression of Amelogenin increased proportional to the number of HERS/ERM cell line at 6
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and 12 week after transplantation (Fig. 4B). These data suggested that the reciprocal interaction between HERS/ERM cell line and SHEDs might be important to induce the
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ACCEPTED MANUSCRIPT 4. DISCUSSION
Epithelial-mesenchymal interaction is involved in the process of not only embryonic
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development but also regeneration process [19]. Tooth is a representative organ, which develops through epithelial-mesenchymal interactions between dental epithelium and dental mesenchyme [3]. As considering the developmental origin of human HERS/ERM cells, it
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may be possible to hypothesize that human HERS/ERM cells might contain or have differentiation potential into ameloblast, enamel-forming cells [20, 21]. To prove this
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hypothesis, we investigated ameloblast-like differentiation of human HERS/ERM cells as a dental epithelial component by co-culturing with SHEDs as a dental mesenchymal component.
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During tooth development, dental epithelium differentiates into ameloblasts through several steps known as the presecretory, secretory, and maturation stages [22]. After crown formation of the tooth is completed, the inner and outer enamel epithelial cells form a bi-
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layered epithelial sheath, called HERS. It has been suggested that HERS cells have an important role in root development [23]. In adults, HERS cells are eventually remained as
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ERM, and some of them disappear by apoptosis [24, 25]. These HERS/ERM cells are a unique population of epithelial cells in the periodontal ligament and are believed to have a crucial role in cementum repair [26]. For the specification of ameloblast-lineage, dental epithelium should interact with dental mesenchyme, which will differentiate into odontoblast. Recently, for the regeneration of teeth, co-transplantation of developing dental epithelium and mesenchyme into kidney capsule could lead to the regeneration of whole teeth [27]. To follow the developmental process of teeth, HERS/ERM cells were co-cultured or co12
ACCEPTED MANUSCRIPT transplanted with SHEDs at 1:1, 2:1, and 3:1 ratio. In previous report, direct co-culture between HERS cells and dental mesenchyme could induce ameloblast-like differentiation [20]. Because the culture medium of HERS/ERM cells SHEDs are different [15, 16], it was
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important to find optimal culture condition to maintain both cell types. Although the size of HERS/ERM cells increased during co-culturing period, they overgrew at 3:1 ratio. On the contrary, in the case of 1:1 ratio, HERS/ERM cells did not proliferate well. The optimal co-
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culturing ratio was 2:1. Because serum-containing growth medium resulted in the cell death of HERS/ERM cells (data not shown), direct contact and various secretory factors derived
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from SHEDs might have beneficial effects on the proliferation and maintenance of HERS/ERM cells during co-culturing period.
After 21 days of co-culture, calcium deposits were observed in both cells and the amount
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of calcium deposits was concentrated at interacting region between HERS/ERM cells and SHEDs. These data also suggested that reciprocal interactions between HERS/ERM cells and SHEDs might be important for the differentiation and calcification. Recently, it was reported
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that mouse HERS/ERM (mHERS/ERM) cells can form mineralized nodules and express enamel- and cementum-related genes [28]. In that report, mHERS/ERM cells were cultured
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on a feeder layer for the maintenance, proliferation, and differentiation. More recently, it was reported that sub-cultured porcine HERS/ERM could differentiate into ameloblast-like cells and generate enamel-like structures in combination with developmental dental mesenchyme [20]. Taken together, these reports suggested the importance of epithelial-mesenchymal interactions for differentiation potentials into ameloblast or odontoblast.
Amelogenin is one of the most important molecules of ameloblast. The basal expression 13
ACCEPTED MANUSCRIPT level of amelogenin in HERS/ERM cells and SHEDs was undetectable; however, the presence or absence of amelogenin is still controversial in HERS/ERM cells. Some articles described the presence of amelogenin [29-31], whereas other studies were not able to detect it
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in HERS/ERM [32-34]. These differences may be explained by cell sources, culture conditions, and detection methods. In our result of co-culturing, the expression of amelogenin increased and localized in the region of HERS/ERM cells not SHEDs. In our results of co-
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of ameloblast-like characteristics of HERS/ERM cells.
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culturing and co-transplantation, the expression of amelogenin was well observed, suggestive
In adult teeth, dental enamel cannot be repaired because there are no cells that synthesize the enamel matrix. Tissue engineering of the enamel organ would provide an important therapeutic option for recovery of enamel loss. In general, tissue engineering involves the
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expansion of specific cell populations in vitro, culturing these cells on a scaffold, and subsequent transplantation of this scaffold into the host. For this purpose, a primary culture of stem cells and ex vivo expansion enough for transplantation are the key prerequisites [35, 36].
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The successful tissue engineering of complex tooth structures will require the characterization of the specific progenitor cell populations that mediate tooth development.
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The potential of ameloblast-like characteristics of HERS/ERM cells are expected to facilitate in vitro enamel regeneration.
ACKNOWLEDGEMENT This research was supported by a grant from National Research Foundation (NRF2013R1A1A1004602 and NRF-2016R1A5A2945889), and Samsung Biomedical Research Institute grant (SMX1161411). 14
ACCEPTED MANUSCRIPT REFERENCE
[1] I. Thesleff, A. Vaahtokari, A.M. Partanen, Regulation of organogenesis. Common
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molecular mechanisms regulating the development of teeth and other organs, Int J Dev Biol, 39 (1995) 35-50.
[2] E. Fuchs, Scratching the surface of skin development, Nature, 445 (2007) 834-842.
SC
[3] I. Thesleff, Epithelial-mesenchymal signalling regulating tooth morphogenesis, Journal of cell science, 116 (2003) 1647-1648.
M AN U
[4] M.J. Honda, S. Tsuchiya, Y. Sumita, H. Sagara, M. Ueda, The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration, Biomaterials, 28 (2007) 680-689.
[5] M. Miura, S. Gronthos, M. Zhao, B. Lu, L.W. Fisher, P.G. Robey, S. Shi, SHED: stem
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cells from human exfoliated deciduous teeth, Proceedings of the National Academy of Sciences of the United States of America, 100 (2003) 5807-5812. [6] S. Gronthos, M. Mankani, J. Brahim, P.G. Robey, S. Shi, Postnatal human dental pulp
EP
stem cells (DPSCs) in vitro and in vivo, Proceedings of the National Academy of Sciences of the United States of America, 97 (2000) 13625-13630.
AC C
[7] W. Beertsen, C.A. McCulloch, J. Sodek, The periodontal ligament: a unique, multifunctional connective tissue, Periodontology 2000, 13 (1997) 20-40. [8] H.F. Thomas, Root formation, The International journal of developmental biology, 39 (1995) 231-237.
[9] B.L. Foster, T.E. Popowics, H.K. Fong, M.J. Somerman, Advances in defining regulators of cementum development and periodontal regeneration, Current topics in developmental biology, 78 (2007) 47-126. 15
ACCEPTED MANUSCRIPT [10] J. Xiong, S. Gronthos, P.M. Bartold, Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues, Periodontology 2000, 63 (2013) 217-233.
periodontal ligament, J Endod, 39 (2013) 582-587.
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[11] D. Keinan, R.E. Cohen, The significance of epithelial rests of Malassez in the
[12] W. Sonoyama, B.M. Seo, T. Yamaza, S. Shi, Human Hertwig's epithelial root sheath cells
SC
play crucial roles in cementum formation, Journal of dental research, 86 (2007) 594-599. [13] J. Xiong, K. Mrozik, S. Gronthos, P.M. Bartold, Epithelial Cell Rests of Malassez
M AN U
Contain Unique Stem Cell Populations Capable of Undergoing Epithelial-Mesenchymal Transition, Stem cells and development, (2012).
[14] T. Tsunematsu, N. Fujiwara, M. Yoshida, Y. Takayama, S. Kujiraoka, G. Qi, M. Kitagawa, T. Kondo, A. Yamada, R. Arakaki, M. Miyauchi, I. Ogawa, Y. Abiko, H. Nikawa, S.
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Murakami, T. Takata, N. Ishimaru, Y. Kudo, Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties, Lab Invest, 96 (2016) 1063-1075. [15] H. Nam, J.H. Kim, J.W. Kim, B.M. Seo, J.C. Park, J.W. Kim, G. Lee, Establishment of
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Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium, Molecules and cells, 37 (2014) 562-567.
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[16] H. Nam, J. Kim, J. Park, J.C. Park, J.W. Kim, B.M. Seo, J.C. Lee, G. Lee, Expression profile of the stem cell markers in human Hertwig's epithelial root sheath/Epithelial rests of Malassez cells, Molecules and cells, 31 (2011) 355-360. [17] J.Y. Lee, H. Nam, Y.J. Park, S.J. Lee, C.P. Chung, S.B. Han, G. Lee, The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells, In vitro cellular & developmental biology. Animal, 47 (2011) 157-164. 16
ACCEPTED MANUSCRIPT [18] H. Nam, G. Lee, Identification of novel epithelial stem cell-like cells in human deciduous dental pulp, Biochemical and biophysical research communications, 386 (2009) 135-139.
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[19] J. Liu, J.J. Mao, L. Chen, Epithelial-mesenchymal interactions as a working concept for oral mucosa regeneration, Tissue Eng Part B Rev, 17 (2011) 25-31.
[20] Y. Shinmura, S. Tsuchiya, K. Hata, M.J. Honda, Quiescent epithelial cell rests of
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Malassez can differentiate into ameloblast-like cells, Journal of cellular physiology, 217 (2008) 728-738.
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[21] M. Athanassiou-Papaefthymiou, P. Papagerakis, S. Papagerakis, Isolation and Characterization of Human Adult Epithelial Stem Cells from the Periodontal Ligament, Journal of dental research, 94 (2015) 1591-1600.
[22] A.G. Fincham, J. Moradian-Oldak, J.P. Simmer, The structural biology of the developing
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dental enamel matrix, J Struct Biol, 126 (1999) 270-299.
[23] J.C. Rincon, W.G. Young, P.M. Bartold, The epithelial cell rests of Malassez--a role in periodontal regeneration?, J Periodontal Res, 41 (2006) 245-252.
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[24] F.M. Wentz, J.P. Weinmann, I. Schour, The prevalence, distribution, and morphologic changes of the epithelial remnants in the molar region of the rat, J Dent Res, 29 (1950) 637-
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646.
[25] H. Kaneko, S. Hashimoto, Y. Enokiya, H. Ogiuchi, M. Shimono, Cell proliferation and death of Hertwig's epithelial root sheath in the rat, Cell Tissue Res, 298 (1999) 95-103. [26] J.D. Spouge, A new look at the rests of Malassez. A review of their embryological origin, anatomy, and possible role in periodontal health and disease, Journal of periodontology, 51 (1980) 437-444. [27] E. Ikeda, R. Morita, K. Nakao, K. Ishida, T. Nakamura, T. Takano-Yamamoto, M. 17
ACCEPTED MANUSCRIPT Ogawa, M. Mizuno, S. Kasugai, T. Tsuji, Fully functional bioengineered tooth replacement as an organ replacement therapy, Proceedings of the National Academy of Sciences of the United States of America, 106 (2009) 13475-13480.
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[28] M. Zeichner-David, K. Oishi, Z. Su, V. Zakartchenko, L.S. Chen, H. Arzate, P. Bringas, Jr., Role of Hertwig's epithelial root sheath cells in tooth root development, Developmental dynamics : an official publication of the American Association of Anatomists, 228 (2003)
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651-663.
[29] T.G. Diekwisch, The developmental biology of cementum, Int J Dev Biol, 45 (2001)
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695-706.
[30] M. Shimonishi, J. Hatakeyama, Y. Sasano, N. Takahashi, M. Komatsu, M. Kikuchi, Mutual induction of noncollagenous bone proteins at the interface between epithelial cells and fibroblasts from human periodontal ligament, J Periodontal Res, 43 (2008) 64-75.
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[31] M. Shimonishi, J. Hatakeyama, Y. Sasano, N. Takahashi, T. Uchida, M. Kikuchi, M. Komatsu, In vitro differentiation of epithelial cells cultured from human periodontal ligament, Journal of periodontal research, 42 (2007) 456-465.
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[32] W. Luo, H.C. Slavkin, M.L. Snead, Cells from Hertwig's epithelial root sheath do not transcribe amelogenin, J Periodontal Res, 26 (1991) 42-47.
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[33] N. Mizuno, H. Shiba, Y. Mouri, W. Xu, S. Kudoh, H. Kawaguchi, H. Kurihara, Characterization of epithelial cells derived from periodontal ligament by gene expression patterns of bone-related and enamel proteins, Cell Biol Int, 29 (2005) 111-117. [34] C.D. Fong, I. Slaby, L. Hammarstrom, Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath, J Bone Miner Res, 11 (1996) 892-898. [35] C.S. Young, S. Terada, J.P. Vacanti, M. Honda, J.D. Bartlett, P.C. Yelick, Tissue engineering of complex tooth structures on biodegradable polymer scaffolds, J Dent Res, 81 18
ACCEPTED MANUSCRIPT (2002) 695-700. [36] B. Hu, A. Nadiri, S. Kuchler-Bopp, F. Perrin-Schmitt, H. Peters, H. Lesot, Tissue
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engineering of tooth crown, root, and periodontium, Tissue Eng, 12 (2006) 2069-2075.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
Figure 1. Primary isolation and characterization of HERS/ERM cells and SHEDs.
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Primarily isolated HERS/ERM cells at passage 3 and SHEDs at passage 6 were characterized. (A) HERS/ERM cells and SHEDs showed epithelial cell-like morphology and fibroblast-like morphology, respectively. (B) The growth curves of HERS/ERM cells and
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SHEDs were compared. (C) The expression of E-cadherin, N-cadherin, and Amelogenin was
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determined by immunofluorescent staining. Magnification was × 200.
Figure 2. Optimization of co-culturing conditions of HERS/ERM cells and SHEDs To find optimal ratio of HERS/ERM cells for co-culture, HERS/ERM cells were co-cultured with SHEDs in three different ratios of 1:1. 2:1, and 3:1 in non-osteogenic medium (A) and
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osteogenic medium (B). The size of HERS/ERM cells increased in proportion to the seeding density regardless of culture conditions. Magnification was × 100. (C) At day 21, cells were stained with Alizarin Red. Calcium deposits were observed in not only HERS/ERM cells but
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also SHEDs. As the ratio of HERS/ERM cells increased, more calcium deposits were
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observed on both cell types. Magnification was × 200.
Figure 3. The expression of E-cadherin and Amelogenin during co-culturing of HERS/ERM cells and SHEDs in vitro To determine ameloblast-like characteristics of HERS/ERM cells, the expression and localization of E-cadherin and Amelogenin was determined by immunofluorescent staining during culture period. (A) When HERS/ERM cells and SHEDs were co-cultured in nonosteogenic condition, the expression of E-cadherin and Amelogenin was co-localized in 20
ACCEPTED MANUSCRIPT HERS/ERM cells. (B) When HERS/ERM cells and SHEDs were co-culture in osteogenic condition, we could observe co-localization of E-cadherin and Amelogenin in HERS/ERM
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cells. Magnification was × 200.
Figure 4. The in vitro calcification and expression of amelogenin after in vivo cotransplantation of HERS/ERM cells and SHEDs
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(A) At day 21 after co-culturing of HERS/ERM cells and SHEDs, calcium deposits were stained with Alizarin Red. Calcium deposits were observed in not only HERS/ERM cells but
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also SHEDs. As the ratio of HERS/ERM cells increased, more calcium deposits were observed on both cell types. Magnification was × 200. (B) HERS/ERM cells and SHEDs were co-transplanted at three ratios (1:1, 2:1, 3:1) into immunodeficient mouse. At 6 and 12 week after transplantation, the expression of amelogenin was observed in the region of
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extracellular matrix and was up-regulated proportional to the number of HERS/ERM cells.
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B
× 400
Growth curve
1000 100 10
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Number of Cells (x106)
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Figure 1.
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Figure 2
2:1
3:1
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7 day
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B HERS/ERM cells : SHEDs
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DAPI
E-cadherin
DAPI
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Day 14
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Amelogenin
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Figure 3
Amelogenin
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Figure 4
A HERS/ERM cells : SHEDs 2:1
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Non-osteogenic medium
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Osteogenic medium
HERS/ERM cells : SHEDs
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ACCEPTED MANUSCRIPT HERS/ERM cells are epithelial remnant of enamel-forming cells. HERS/ERM cells have ameloblast-like characteristics via interaction with SHEDs. For tooth regeneration, both HERS/ERM cells and SHEDs will be necessary for epithelial-
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mesenchymal interaction.