Visualization of junctional epithelial cell replacement by oral gingival epithelial cells over a life time and after gingivectomy

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)–positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin β4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.


Regenerated JE cells after gingivectomy originate from OGE cells. Currently, it is believed that
the JE regenerates itself when it suffers a minor injury, while it is regenerated by OGE when largely removed by gingivectomy 28 . However, it has not been possible to completely exclude the possibility that remnants of the JE contribute to regeneration after gingivectomy. The tooth germ transplantation technique is a useful method to eliminate this possibility because if remnants of the JE proliferate and regenerate, the regenerated JE should be GFP-positive. Therefore, using this technique, it was determined whether JE derived from the odontogenic epithelium has the ability to regenerate the JE after partial gingivectomy. One day after gingivectomy, no GFP fluorescence was observed at the gingivectomy site (dotted line in Fig. 4A). Seven days after gingivectomy, the surgical site was covered with epithelium just like before the gingivectomy. Green fluorescence was not detected in the gingiva at the surgical site on days 7 and 14, although fluorescence was detected in the gingiva at the opposite side, where gingivectomy had not been performed (Fig. 4A). Histological analyses were performed 14 days after gingivectomy. GFP-positive cells were detected in the control JE, but not in the regenerated JE (Fig. 4B,C). In addition, to examine whether normal adhesion of the regenerated JE recovered, we examined the expression of Itgb4 and Lama5 in the regenerated JE and the control JE. In the regenerated JE, Itgb4 was detected in the IBL and EBL and close to the basal JE cells (white arrowheads in Fig. 4D). Lama5 was also detected in the IBL and EBL of the regenerated JE (white arrowheads in Fig. 4E). Therefore, we were able to verify the normal adhesion of the regenerated JE to the enamel surface through adhesion molecules consisting of hemidesmosomes (Fig. 4D,E). These results suggest that the regenerated JE after partial gingivectomy arose not from residual JE cells but rather from OGE.
Characterization of odontogenic epithelium-derived JE and OGE-derived JE. Based on analysis of tissue after partial gingivectomy, regenerated JE was found to be derived from OGE rather than odontogenic epithelium. To precisely characterize the odontogenic epithelium-derived JE and OGE-derived JE, we compared the gene expression profiles between odontogenic epithelium-derived JE dissected from mice with GFP-positive tooth germ transplantation, OGE-derived JE dissected from mice 30 days after gingivectomy, and OGE from the palatal region via RNA sequencing (Table S1 and Data S1). Through hierarchical clustering analysis, the odontogenic epithelium-derived JE and the OGE-derived JE were grouped into the same cluster, but the palatal OGE was not (Fig. 5A). On the other hand, based on principal component analysis (PCA), the gene www.nature.com/scientificreports www.nature.com/scientificreports/ expression patterns of the odontogenic epithelium-derived JE, OGE-derived JE, and palatal OGE were dissimilar (Fig. 5B). Interestingly, several JE-specific genes such as odontogenic genes, ameloblast-associated (Odam), intercellular adhesion molecule 1 (Icam1), S100 calcium-binding protein A8 (S100a8), and S100 calcium-binding protein A9 (S100a9) were commonly upregulated in odontogenic epithelium-derived JE and OGE-derived JE relative to OGE (Fig. 5C). Especially Odam, the expression in OGE-derived JE cells was higher than odontogenic epithelium-derived JE cells. Next, to analyze the difference between odontogenic epithelium-derived JE and OGE-derived JE, we conducted comprehensive analysis of profiling data using the Venn diagrams and Gene Ontology (GO) enrichment analysis. The Venn diagram revealed that 250 genes are commonly upregulated in OGE-and odontogenic epithelium-derived JE cells compared to OGE. In contrast, 404 genes were expressed in OGE-derived JE cells but not in odontogenic epithelium-derived JE cells, while 43 genes were specific only to odontogenic epithelium-derived JE cells (Fig. 5D). GO enrichment analysis demonstrated that some OGE-derived JE cell-specific genes are associated with inflammatory response and chemotaxis (Data S2). www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the present study, we directly revealed that JE derived from the odontogenic epithelium was replaced by OGE cells over time. In addition, JE that regenerated after gingivectomy originated from OGE cells. OGE-derived JE expressed JE-specific genes, but did not have the same gene expression profile as odontogenic epithelium-derived JE.
Periodontal disease is caused by bacteria found in dental plaque and many other factors, including local and systemic immunoinflammatory responses and environmental factors 8,11 . JE attaches to the tooth surface and  www.nature.com/scientificreports www.nature.com/scientificreports/ forms a defensive line against periodontal bacterial infection. The JE is a non-keratinized epithelium and has wide intercellular spaces that are easily infiltrated by inflammatory cells such as neutrophils and monocytes. Thus, the specific features of the JE are expected to be important barriers against bacterial infections. Therefore, functional changes to the JE may affect the progression of periodontal diseases. Importantly, based on previous histological analyses, the primary JE is believed to be formed by the fusion of the reduced enamel epithelium with the oral epithelium and is gradually replaced by the oral epithelium, suggesting that the functions of the JE might change over the course of a lifetime. To date, studies have shown only indirect evidence for this because there have been no tracing methods to identify the origins of the JE. In the present study, we directly determined the origin of the JE using a tooth germ transplantation technique. JE turnover is reported to be 4-6 days based on the mitotic index and radiography in marmosets and monkeys 13,29 . Furthermore, BrdU-positive cells have been detected in the basal cells of the JE after 2 h and are more distinct after 48 h 30 . This means that the JE has the potential to self-renew. Consistent with this, our previous report also demonstrates the same proliferative potential of the JE, suggesting that the JE generated by our technique is an appropriate model for evaluating the characteristics of normal JE.
In the present results, the JE derived from odontogenic cells was indeed replaced gradually by OGE cells from the basal layer over the course of the 140 days after transplantation of GFP-positive tooth germs. Consequently, JE derived from odontogenic epithelium was mostly replaced by OGE cells after 200 days. Consistent with this, when GFP-positive tooth germs were transplanted into the alveolar bone in ROSA mT/mG mice, similar results were obtained. Therefore, we concluded that odontogenic epithelium-derived JE maintained the potential for self-renewal for some time before finally losing it in part or entirely. On the contrary, in the lingual epithelium, multicolor lineage tracing has demonstrated that one stem cell per interpapillary pit survives long-term 31 . The replacement of odontogenic epithelium-derived JE with OGE may be driven by several mechanisms, such as loss of the self-renewal potential of the odontogenic epithelium-derived JE due to aging or cell competition between odontogenic epithelium-derived JE cells and OGE cells, although the exact mechanisms remain unclear. Therefore, further investigation is necessary in the near future.
There are many reports of restoration after gingival resection [32][33][34][35][36] . In addition, Masaoka et al. reported no obvious morphologic changes in the JE between sites subjected to gingivectomy and control sites and the expression of Itgb4 and Lama5 in regenerated gingiva 14 days after mouse gingival resection 37 . These reports do not show the origin of repaired cells. Our study of gingival resection of tooth germ transplantation directly determined that the origin of regenerated JE was OGE cells. Therefore, we are able to confirm that remnants of odontogenic epithelium-derived JE did not contribute to regeneration of the JE after gingivectomy, which was previously unclear. Furthermore, regenerated JE began to express Itgb4 and Lama5. Regenerated JE showed a similar structure to the odontogenic epithelium-derived JE. Additionally, the regenerated JE was considered to have acquired a normal structure. Several studies have reported that cells directly attached to the teeth (DAT cells) are involved in the adhesion of the enamel surface layer [38][39][40] . In electron microscopy analyses, DAT cells have been found to have numerous microvilli-like structures on the cell surface, which firmly adhere to the tooth surface and migrate DAT cells from the apical to the coronal side. In addition, Itgb4 expression is involved in DAT cell adhesion and migration as well as epithelial turnover 38 . In the present study, migration of DAT cells was not detected at the resected site, and it was revealed that recovery of odontogenic epithelium-derived JE did not occur after gingivectomy.
To characterize JE derived from different origins, we compared the gene expression profiles of the odontogenic epithelium-derived JE and OGE-derived JE, which were resected from transplanted teeth and regenerated JE, via RNA sequencing. Surprisingly, several genes, such as Odam, Icam1, S100a8, and S100a9, were commonly upregulated in odontogenic epithelium-derived JE and OGE-derived JE relative to OGE. Importantly, Odam, an ameloblast-associated gene, was found to be a common upregulated gene in OGE-and odontogenic epithelium-derived JE cells. Odam is also reported to be specific to JE cells 33 . Therefore, the OGE might have the potential to partly acquire the characteristics of odontogenic epithelium-derived JE. However, it was unexpected that the Odam expression in OGE-derived JE cells was much higher than that in odontogenic epithelium-derived JE cells. The Odam-ARHGEF5-RhoA signaling pathway is reported to play a significant role in tooth-cell adhesion. Moreover, laminin, which activates integrin-mediated Odam signaling, participates in proliferation, differentiation, and actin rearrangement during JE formation [41][42][43][44][45] . We speculate that the high Odam expression in the replaced JE might be due to proliferation, differentiation, and actin rearrangement during OGE-derived JE formation after gingivectomy. Consistent with this, GO enrichment analysis demonstrated that some OGE-derived JE cell-specific genes are associated with inflammatory response and chemotaxis. Therefore, these data suggested the possibility that OGE-derived JE cells were still in the process of wound healing, although 30 days after gingivectomy, OGE-derived JE cells appear morphologically normal. PCA revealed that the gene expression patterns of odontogenic epithelium-derived JE and OGE-derived JE were not similar. Therefore, it is an important thing to clarify whether gene expression patterns of OGE-derived JE gradually become similar to those of odontogenic epithelium-derived JE or not. This will be examined in future studies.
(Tokyo, Japan). C57BL/6-KI (ROSA mT/mG ) mice were purchased from The Jackson Laboratory 47 . All mice were born alive and maintained under specific pathogen-free (SPF) conditions. The mouse experiments were approved by and conducted according to the guidelines of the Showa University Animal Care and Use Committee (number 18007).
GFP-positive tooth germ transplantation method. The tooth germ transplantation method was performed as previously described 48 . Briefly, molar tooth germs were collected from the mandibles of C57BL/6-Tg (CAG-EGFP) mice on E14. The collected molar tooth germs were placed in a gel drop of Cellmatrix type I-A www.nature.com/scientificreports www.nature.com/scientificreports/ (Nitta Gelatin). Then, the tooth germs were cultured in Dulbecco's modified Eagle's medium (DMEM) (Wako) Supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere at 5% CO 2 for 4 d. The upper first molars of 3-week-old C57BL/6 N (WT) and C57BL/6-Tg (ROSA mT/mG ) mice were extracted under deep anesthesia and then the alveolar bones at the tooth extraction sites were allowed to heal for 3 weeks. A hole approximately 1.0 mm in diameter was made in the exposed alveolar bone surface of C57BL/6 N and ROSA mT/mG mice and the tooth germ was transplanted into the hole.
Histological analyses after gingivectomy. The palatal gingiva of the transplanted teeth of WT mice were excised using a surgical knife (No. 11; Feather, Osaka, Japan) under deep anesthesia. The buccal side served as a control. A piece of gingival tissue including GFP-positive JE was resected from the medial to the distal side of the transplanted tooth according to a method described in a previous study 37 . The gingival tissue around the transplanted teeth was observed using a fluorescence stereomicroscope on days 0, 1, 7, and 14 after gingivectomy (SZX7; Olympus, Tokyo, Japan). Histological analyses of the JE around the transplanted teeth in WT mice were performed 14 d after gingivectomy.
Micro-CT scanning. Scanning was performed using a micro-CT device in an in vivo 3D µCT system according to the manufacturer's protocol (R_mCT2, Rigaku Co., Ltd., Tokyo, Japan).
Immunohistological analyses. The maxillae were dissected and fixed with 4% paraformaldehyde for 6 h at 4 °C after decalcification with 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks at 4 °C. The specimens were embedded in optimal cutting temperature compound (Sakura) and then immediately snap-frozen in liquid nitrogen-cooled isopentane. The frozen sections were cut using a cryomicrotome (Microm) to 5 μm thickness in the buccal-lingual direction. Hematoxylin and eosin (H&E) or immunofluorescence staining were performed on the sections. For immunofluorescence staining, the frozen sections were air-dried for 10 min, washed with tris-buffered saline (TBS), and pre-incubated with blocking solution (Dako) for 10 min. The sections were incubated with an anti-integrin β4 rat polyclonal antibody (Cat. No. ab25254; 1:100: Abcam) for 1 h and an anti-laminin 5 rabbit monoclonal antibody (Cat. No. ab14509; 1:200: Abcam) for 2 h at room temperature. After washing in TBS, the sections were incubated for 30 min at room temperature with an anti-rabbit IgG antibody conjugated with Alexa 594 or an anti-rat IgG Alexa 594 of donkey origin (1:200 dilution; Molecular Probes). After counterstaining with 49, 6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:500 dilution; Dojindo), all specimens were examined and photographed (BZ-9000 fluorescence microscope, Keyence).

RNA sequencing.
Odontogenic epithelium-derived JE was collected from the GFP-positive gingiva around the transplanted teeth. Odontogenic epithelium-derived JE and OGE-derived JE were collected from GFP-positive tooth-transplanted WT mice using a surgical knife (No. 11; Feather, Osaka, Japan) with a stereomicroscope under deep anesthesia. The odontogenic epithelium-derived JE was resected as a GFP-positive gingiva surrounding the crown of the transplanted tooth. The OGE-derived JE collected the regenerative region around the transplanted tooth 30 days after gingivectomy, while the OGE collected the gingiva distant from the junctional epithelium. Under the stereomicroscope, mesenchymal tissue was surgically removed as much as possible (n = 2 mice per group. One specimen for one biological replicate.).
OGE-derived JE was resected from GFP-negative regenerated JE on day 30 after gingivectomy. Non-junctional OGE was collected from palatal gingiva. Total RNA was extracted from tissue using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. A library for RNA sequencing was prepared using the TruSeq ChIP Sample Prep Kit according to the manufacturer's instructions. Paired-end sequencing (read length: 101 + 101) was carried out using the Illumina HiSeq 2500 system. The sequence reads were aligned to the mouse reference genome (mm10) using Tophat 2.0.13 (bowtie2-2.2.3), which can adequately align reads to the location, including splice sites, in the genome sequence. Sequence data were analyzed using HCS version 2.2.58, RTA version 1.18.64, bcl2fastq-1.8.3, and CLC Genomics Workbench.

Reverse transcription PCR (RT-PCR).
Total RNA was extracted from tissue samples using the RNeasy Mini Kit Plus (Qiagen) according to the manufacturer's instructions (n = 3 biological replicate). Complementary DNA (cDNA) was generated by reverse transcription using SuperScript VILO Master Mix (Thermo Fisher).
The cDNA was mixed with Power SYBR ® Green PCR Master Mix (Thermo Fisher) and specific gene primers.
PCR was performed using the Gene Amp PCR System 9700 (Thermo Fisher) with the primer sequences listed below. The amplification conditions consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing and elongation at 60 °C for 1 min. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an endogenous control.