Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels

archives of oral biology 56 (2011) 1199–1220

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Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels Margaret R. Byers a,*, Ruth E. Westenbroek b a b

Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, WA 98195, United States Department of Pharmacology, University of Washington, Seattle, WA 98195, United States

article info

abstract

Article history:

Objective: Our goal was to evaluate the expression patterns for voltage gated sodium

Accepted 21 April 2011

channels in odontoblasts of developing and mature rat teeth. Design: We analysed immunoreactivity (IR) of the alpha subunit for all nine voltage gated

Keywords:

sodium channels (Nav1.1–1.9) in teeth of immature (4 weeks), young adult (7 weeks), fully

Non-neuronal

mature adult (3 months), and old rats (6–12 months). We were interested in developmental

Nav1.1–Nav1.9

changes, crown/root differences, tetrodotoxin sensitivity or resistance, co-localization with

Dental pulp

nerve regions, occurrence in periodontium, and coincidence with other expression patterns

Gingiva

by odontoblasts such as for transient receptor potential A1 (TRPA1).

Periodontium

Results: We found that Nav1.1–1.9-IR each had unique odontoblast patterns in mature

TRPA1

molars that all differed from developmental stages and from incisors. Nav1.4- and

Nestin

Nav1.7-IR were intense in immature odontoblasts, becoming limited to specific zones in adults. Crown odontoblasts lost Nav1.7-IR and gained Nav1.8-IR where dentine became innervated. Odontoblast staining for Nav1.1- and Nav1.5-IR increased in crown with age but decreased in roots. Nav1.9-IR was especially intense in regularly scattered odontoblasts. Two tetrodotoxin-resistant isoforms (Nav1.5, Nav1.8) had strong expression in odontoblasts near dentinal innervation zones. Nav1.6-IR was concentrated at intercusp and cervical odontoblasts in adults as was TRPA1-IR. Nav1.3-IR gradually became intense in all odontoblasts during development except where dentinal innervation was dense. Conclusions: All nine voltage-gated sodium channels could be expressed by odontoblasts, depending on intradental location and tooth maturity. Our data reveal much greater complexity and niche-specific specialization for odontoblasts than previously demonstrated, with implications for tooth sensitivity. # 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Odontoblasts are unusually complicated cells that differentiate from cranial neural crest, as is true for many cranio-facial tissues.1–3 After odontoblasts leave the cell cycle, they pass through many well-defined stages of maturation4,5 and a

succession of transcriptomes6 as they first build primary dentine, followed by secondary dentine, a mature pulp chamber and ongoing reactions to tooth wear. Odontoblasts are postmitotic for many decades, they attract specialized vascular and neural support,7–10 and they have unusual gene expression.9–14 Odontoblast functions include dentinogenesis plus maintenance/repair of dentine in adults,14,15 and pulp/dentine barrier

* Corresponding author. Tel.: +1 206 543 8629; fax: +1 206 543 3079. E-mail addresses: [email protected] (M.R. Byers), [email protected] (R.E. Westenbroek). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.04.014

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regulation.16,17 In addition they have primary cilia enabling their mechanosensitivity,18 they express a variety of voltageand ligand-gated ion channels,11,12,19–23 and they move when tooth stimuli cause fluid movement in dentine and sharp pain.24–26 Ultrastructural studies have revealed many details of odontoblast polarity, intradentinal process structure, and numerous interconnections via a terminal web and gap junctions27–37 that enable odontoblasts to behave as an epithelioid syncytium,38 and to have gap junction connexions with nearby pulp cells but not nerve fibres.35–38 Recently odontoblasts have also been shown to have specific differential gene expression for innate immune responses in organ culture39–41 and other immune capabilities as defined with in vitro models.42,43 In order to do all the diverse functions noted above, odontoblasts need to have complex abilities to sense their environment, communicate amongst themselves and with nearby cells, and coordinate morphogenic and mature responses of the odontoblast layer. Voltage gated sodium channels are amongst the key tools that enable cells to respond effectively to their environment,44,45 and here we explore expression of all nine Nav isoforms in odontoblasts of mature rat molars compared with developmental stages. There is much interest in understanding the purpose of these channels in odontoblasts that might indicate direct actions by odontoblasts during initiation of nerve activity in teeth (and hence for dental pain). Alternatively, the rich array of neural-like channels in odontoblasts may be for local coordination of dentinogenic responses to the gradual (or acute) attrition of enamel and dentine during decades of chewing, invasion by caries, or injury. The close proximity of nerve fibres and odontoblasts in coronal pulp horn regions includes neural-odontoblast apposition junctions near the terminal web and thin nerve fibres in as many as half of the dentinal tubules beneath cusp tips.27–35 In addition, many excitable neural-type membrane channels have been found in immature human pulp cells or odontoblasts in vitro11,12,19–22,46,47 or in immature and adult teeth23,48,49 and odontoblasts can make action potentials under some circumstances.12,19,21,49 However, other craniofacial tissues derived from neural crest such as pulpal fibroblasts23,50 and alveolar osteoblasts51,52 also have complex voltage-gated signalling that may be entirely for local tissue homeostatic responses to environmental stimuli. In addition, the voltagegated channels in odontoblasts may enable glial-like activity23 to support the sensory nerve endings that extend beyond the terminal glia in pulp. It should be noted that the dental neural apparatus has its own specific receptors that include mechanosensitive channels,53 rapidly adapting (vibration sensitive) mechanosensory neurons that conduct at high velocity along trigeminal nerves,54–58 many slower polymodal, purinergic, or specific nociceptors, and osmotic or thermosensitive nerve fibres.53,59–61 These could give the dental innervation sufficient capability by itself to enable the hydrodynamic transduction mechanisms implied by human studies that elicit fluid movement in dentine,62–65 especially since the rapidly adapting mechanoreceptors respond too quickly for the direct involvement of any other cell.54 In relation to dental pain mechanisms, it is noteworthy that neural Nav1.7-IR,66 pulpal Nav1.8 protein,67 and neural Nav1.9-IR61 can increase in painful human teeth and in other pathologic pains.68

Very likely most or all of the neuro–pulpal interactions that have been suggested can occur in some part(s) of the tooth, or under certain conditions or maturation phases, as suggested for four likely mechanisms that encompass mechanoreceptive and nociceptive events by Gunji.29 The demonstration of purinergic neurons in fully formed human teeth60 adds a specific paracrine mechanism by which adenosine triphosphate (ATP) released from mature odontoblasts or other pulp cells might activate some dental nerves, as also suggested by the many changes in tooth sensitivity when pulpal conditions change.69,70 Most of the information about ion channels in odontoblasts has come from in vitro analyses of immature samples (odontoblasts from extracted unerupted human third molar teeth) that are then grown in culture to produce odontoblast-like features such as expression of dentine sialoprotein (DSPP),71 or from perpetually immature rodent incisors.6,21 That approach has identified important signalling capabilities of odontoblasts.19–23,46–49 However, the very different functions and gene expression profiles for adult permanent teeth compared with immature teeth6,36,72 show the need to study dental Nav channels in much greater detail in adults. Our goal here was to determine whether the distribution of Nav isoforms in immature and mature teeth is informative about shifting odontoblast activities during tooth development and mature functions. Accordingly, we have surveyed the staining patterns for all nine alpha subunits of sodium channels in rat teeth. Cellular activation via voltage-gated sodium channels (Nav1.1–1.9) drives propagated signals in neurons and muscle cells as well as more localized signals in most other cells,44,45,73–75 with each mature tissue having its own specific Nav isoform combinations that differ from the temporary set that typifies immature stages.76–78 We asked how those patterns change during development of rat molar teeth, and whether there are specialized micro environments (niches) with distinctive expression, as found recently for other odontoblast-regulatory signals.79 In this study we focused on the odontoblasts of cusp tip, anterior and posterior sides of pulp horns, intercusp, cervical crown, furcation, and within the roots. Our hypotheses were that (1) sodium channel isoforms Nav1.1–1.9 each have unique expression patterns in odontoblasts of mature molars that differ from developing teeth, (2) mature patterns are maintained in odontoblasts during pulp constriction in ageing molars, (3) some odontoblast Nav patterns are related to terminal distribution of the dentinal innervation, (4) reparative dentinogenesis requires special Nav patterns, (5) some odontoblast Nav isoforms will colocalize with the pain-related transient receptor potential TRPA1,22,80 (6) patterns for tetrodotoxin-sensitive channels will share some features and differ from tetrodotoxinresistant channels, and (7) the spatial patterns of Nav expression in gingiva and other periodontal tissues will coincide with some of the odontoblast spatial patterns. Our approach has been comparative immunohistochemistry (IHC) of tissue sections because of the importance of defining exact positions within the odontoblast layer for specific sodium channel phenotypes. The results are discussed in relation to differences between immature and mature teeth, variety of odontoblast phenotypes, voltage-gated sodium channel functions in neurons and non-neuronal cells, odontoblast–nerve

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interactions, crown–root differences, and sensory mechanisms in teeth.

2.

Materials and methods

2.1.

Animals

These studies were approved by the Institutional Animal Care and Use Committee of the University of Washington. We studied molars at four different ages: immature at 4 weeks (with initial crown and root morphogenesis partly completed), young adults at 7 weeks (with newly closed root apices), fully mature adults at 3 months, and old rats. At each stage, the Nav patterns of expression were analysed in at least three different rats, in both maxillary and mandibular jaws. We always included multiple ages from simultaneous fixation, decalcification and sectioning groups in each immunohistochemical run, as well as more than one antibody plus positive and negative controls. Total number of rats was 75, including four week (N: 17), seven week (N: 21), three month (N: 32), and 6–12 month old rats (N: 5).

2.2.

fixation, duration of decalcification, sectioning thickness, and timing between sectioning and IHC. In many cases, samples from old adults (6–12 months) were also included. Floating sections were reacted in primary polyclonal antibody diluted in 0.1 M sodium phosphate buffer plus 2.5% normal goat serum and 2.5% rat serum (Sigma–Aldrich, St. Louis. MO). Bound polyclonal antibodies were detected by incubation in biotinylated goat anti-rabbit IgG (1:500 dilution, Vector, Burlingham, CA), followed by the peroxidase avidin/biotin (ABC) reaction (Vector) and diaminobenzedine (DAB; Sigma– Aldrich, St. Louis, MO) plus H2O2. Monoclonal primary antibodies: anti-Nav1.4 (Sigma–Aldrich) and anti-nestin (Millipore) were diluted 1:500 in buffer plus 2.5% horse serum (Sigma–Aldrich), with control sections lacking monoclonal antibody. Detection was via biotinylated horse anti-mouse IgG (Vector) at 1:500 dilution followed by reaction with ABC (Vector) and diaminobenzedine plus H2O2. Most sections were mounted with pale or absent counterstain, but in some cases there was stronger counterstain (Methylene Blue). Sections were visualized using a Nikon light microscope. Digital images had similar adjustment to grayscale and framing of the intensity by removal of the empty high and low levels (Adobe Photoshop), without any other changes in brightness or contrast.

Tissue preparation 2.4.

Rats were deeply anesthetized with sodium pentobarbital diluted in sterile saline (75–80 mg/kg) and then fixed by transcardiac perfusion with 4% paraformaldehyde. Additional fixation of dissected jaws was kept brief (2–4 h) to facilitate detection of sodium channels by immunohistochemistry. Fixed jaws were decalcified in 10% EDTA (pH 7.4) with solution changes 2–4 times per week, and equilibrated in 30% sucrose in 0.1 M sodium phosphate (PO4) buffer for several days prior to cutting 40 mm thick frozen sections in the anterior/posterior longitudinal plane to show all three molars per jaw. Mandibular incisors were included in the mandibular jaw samples. For each antibody, we reacted evenly spaced sections (intersection interval: 120–160 mm). This yielded 3–5 sections per jaw per antibody (2–4 antibodies per jaw) each providing full views of crown and root pulp.

2.3.

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Diaminobenzendine immunohistochemistry

Polyclonal antibody specificity: Anti-Nav1.1–1.3, 1.5–1.9 (Alomone, Jerusalem, Israel) and anti-Nav1.3 and -Nav1.6 (Millipore, Temecula, CA) were affinity purified antibodies against specific epitopes (accession numbers and western blot preadsorption data at: http://www.Alomone.com; http:// www.Millipore.com). The polyclonal anti-Nav antibodies were reacted at 1:500–1:2000. Other polyclonal antibodies were anticalcitonin gene-related peptide (Sigma–Aldrich, 1:3300), antiperipherin (Millipore, 1:1000) and anti-TRPA1 (Alomone, 1:1000). Technical controls included pre-adsorption of primary antibody with antigen, omission of primary or secondary antibodies, as well as the consistently different patterns that we found for different Nav isoforms within the same IHC run, and the positive control tissues near the teeth. For each immunohistochemical experiment, we compared two or more anti-Nav antibodies and included jaw samples from immature, newly mature, and young adult ages that had similar

Immunofluorescence

Jaw sections of young adults were double labelled using previously published methods.23 The polyclonal Nav 1.8 primary antibody (1:100 dilution; Alomone, Jerusalem, Israel) was visualized using biotinylated goat anti-rabbit second antibody (1:300 dilution, Vector) and avidin D-fluorescein (1:300; Vector). The monoclonal antibody for calcitonin generelated peptide (CGRP; 1:750 dilution, Sigma) was visualized using goat anti-mouse Alexa 555 (1:1000; Invitrogen, Eugene, Oregon). Digital images were collected using a Leica SL confocal microscope (WM Keck Imaging Facility, Univ. Washington).

2.5.

Analyses and quantitation

We initially viewed DAB-stained patterns for all nine Nav isoforms in a Nikon light microscope, and recorded key findings by digital photography without contrast enhancement. The diagram for Fig. 4 was based on measurements of several mandibular first molar samples at each age. We then made a qualitative comparison of the Nav patterns at the four ages. Our qualitative scale (Table 1) registered five degrees of intensity [none, weak (A), moderate (B), strong (C), or intense (D)] for the odontoblasts in each of the eight odontoblast phenotype zones (Fig. 5) at three ages. The evaluated samples all had excellent immunohistochemistry, as shown by positive control sites (Fig. 6). Median values are shown (Table 1) for each Nav isoform/per intradental zone/per age group (4 week, 7week, 3 months). Quantitative comparisons of odontoblast immunostaining for key Nav isoforms were also made for adult molars by specific densitometry (Table 2). Digital (TIF) images were inverted in photoshop (Adobe), and then were imported into Igor (Wavemetrics). Using the Image Processing package in this software program, we outlined the tissue area of interest

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Table 1 – Intensity of Nav isoform immunoreactivity in odontoblasts per zone.a Nav1.4

Nav1.7

Nav1.1

Nav1.5

Nav1.3

Nav1.8

Nav1.6

Nav1.9

Nav1.2

Zone 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m 4w 7w 3m I II III IV V VI VII VIII Ging Trend

B C A A A B A A D C D C C A B B 5 5 CR & RT

C A – – A – – B 5 +

A – B A B B C A B A C B C A D B 2 2 CR & RT

– A A A A B A B 2 +

– – A A A A A B B A A B B C B B C B B B A B B B 1 1 1 CR * & RT +

B B B B C B C B C B B B B C B C C B C D A C C C 3 3 3 CR * & RT +

A B A A – A A B A A B B B B C C 4 4 CR & RT

C A B C C D D D 4 *

– – A B B C A B A B A A – – – – 1 1 CR *

A C C B B A – A 1

– – – – – A – – B A A B B A A B A A A A B B A A A A A B B B A A A B B A B B A B – – A B B A B B A A – B A A B B A A A A A A A A 1 1 1 1 1 1 3 3 Special focal sites in Adult molars

– B A A A A A A 3

a Our qualitative scale registered five degrees of intensity: none (–), weak (A), moderate (B), strong (C), or intense (D) for odontoblasts in each of the eight zones. Dashed boxes show sites of comparative densitometry (Table 2) for levels III, IV at Nav1.5, Nav1.3, Nav1.8. Gingiva (Ging) categories 1–5 in text and Fig. 6. Trend: Developmental Trend. CR: crown, RT: root.

Table 2 – Pixel intensity for Zones III and IV in adult rat molars.a Isoform Nav1.3 Nav1.5 Nav1.8

Zone III (N)

Zone IV (N)

p value

73.3  14.23 (8) 89.3  11.20 (5) 107.2  6.10 (7)

121.9  15.01(8) 47.5  7.36 (5) 62.2  12.30 (7)

<0.05 <0.02 <0.01

Immunohistochemistry groups were compared using the same pixel intensity range (0–250) and Igor software (Wavemetrics). N: 5– 8 rats per Nav as shown. Significance: unpaired Student’s t-test to compare Zone III with Zone IV per Nav isoform. a Mean  standard error.

stained by immunocytochemical methods and then used the Region of Interest function (ROI) in Igor to determine the average pixel intensity of the area of interest. In that analysis we focused on the pulp horn and compared the odontoblast layer on the well-innervated side (Zone III), that is found along anterior side of maxillary pulp horns and posterior side of mandibular pulp horns,81 with the less innervated side (Zone IV). We report the average pixel intensity  standard error of the mean. Several intensity readings were made per section per Zone for Nav1.3-IR, Nav1.5-IR, and Nav1.8-IR. One or two

jaws were measured per rat (all averaged to give one value per rat per Nav). Significance was at the level of p < 0.05 (Students t-test, unpaired, two-tailed).

3.

Results

3.1. Odontoblast expression of Nav isoforms is related to pulpal and dentinal specializations The data in this paper come from multi-cusped rat molars, for which crown structure, cusp orientation and occlusal contacts are shown for mature adults (Fig. 1). The Nav isoform-specific patterns in molars had opposite orientations related to whether cusps tilted towards posterior (maxillary) or anterior (mandibular) directions. The expression patterns of Nav proteins in molars of fully mature adult rats (3 months old) revealed crown/root differences and varied expression foci depending on the types of dentine, the pulpal sub-region, and the specific Nav isoform investigated (Fig. 2). This is shown at low magnification for Nav1.8 in the mandibular first molar posterior crown/root compared with Nav1.3, Nav1.4, Nav1.7, and Nav1.9 (Fig. 2A–E). The coronal asymmetric distribution of

Fig. 1 – Rat molar cusp orientation and occlusal surface shape. Maxillary (AB) and mandibular (CD) molars of rats contain multiple cusps, with an opposite anterior–posterior (A–P) orientation for maxillary compared to mandibular molars. The maxillary cusps have a convex occlusal centre (B), whilst mandibular molar cusps are concave (D). Jaws for this study were serial sectioned in the mesio-distal longitudinal plane to show all three molars (1–3) per section. Scale: 0.5 mm.

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Fig. 2 – Variety of odontoblast patterns. Nav1.1–1.9 alpha subunits each had a specific pattern of expression, shown here in fully mature molars for Nav1.8 (A), Nav1.3 (B), Nav1.4 (C), Nav1.7 (D), and Nav1.9 (E), compared with asymmetric distribution of nerve fibres containing calcitonin gene-related peptide (CGRP) (F, arrows). There were three crown regions: the special tip (*); the pulp horn (PH); and cervical pulp (C). The furcation between roots (F) faces interradicular ligament, and its odontoblast phenotypes were similar to nearby root zones that comprised pulp near the crown (R1), a middle zone (R2), and at the apical root zone (R3) that passes through cellular cementum (cc). Odontoblasts had distinctive label, but pulpal dendritic cells had Nav1.9-immunoreactivity (IR), and nerve fibres had Nav1.3-, Nav1.7- and Nav1.8-IR. Pulp staining differed from periodontal ligament (pdl) and alveolar bone (bone). Nav1.7 focuses in root odontoblasts (D, arrows) of adult. Dots: region of crown that attracts the most innervation (A,B,D,E,F: mandibular molars, C: maxillary molar). N: Nav. Scales: A, B: 0.5 mm; C–F: 0.2 mm.

CGRP-IR nerve fibres is also shown (Fig. 2F). The patterns for Nav concentrations in mature adults (Fig. 2) were compared with molars of year-old rats for Nav1.8 and Nav1.4 (Fig. 3). We found that their inverse patterns that avoid (Nav1.8) or associate with (Nav1.4) the special tip dentine and reparative dentine were maintained as teeth were worn down during ageing. The molar patterns for Nav1.8-IR and Nav1.4-IR differed in old adults from the continuously-erupting incisor teeth whose staining patterns remained immature in those same jaws (Fig. 3A and E).

3.2.

Pulpal micro environments defined

The Nav patterns of localization defined four general odontoblast-lined pulpal regions in the crown and two in the root for young and old molars (Figs. 2–4). These pulp zones were

defined by: (1) the special tip dentine (tip) that is not covered by enamel; (2) the enamel-covered pulp horn (PH) dentine, where most of the innervation terminates; (3) the enamel-covered cervical crown chamber (C) comprising intercusp regions and the sides of the main crown chamber; (4) the furcation (F) on the floor of the crown, with its dentine facing towards the interradicular periodontal ligament and alveolar bone, and (5– 6) two odontoblast-lined root (R) regions, R1 near the crown, and R2 between R1 and R3. The R3 root tip connects pulp and periodontium via neurovascular channels that are lined by cellular cementum and so do not have odontoblasts. The pulp horn region was further subdivided into three different Nav isoform domains, as indicated (Fig. 5), giving a total of at least eight different phenotypes of odontoblasts in adults based on their expression of Nav isoforms. These regions were: Zone I (tip); Zone II (adjacent to tip); Zones III and IV (sides of pulp

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horn); Zone V (cervical pulp); Zone VI (interdental facing side of root); Zone VII (interradicular facing side of root); and Zone VIII (furcation region). Zone III is the side of the pulp horn with the greatest innervation, as defined earlier81 and was located on the anterior side of maxillary pulp horns and posterior side of mandibular pulp horns. Zone IV has been shown to have less dense innervation than Zone III.

3.3.

Fig. 3 – Preservation of pulpal Nav expression patterns in old teeth. (A) One year-old rat stained for Nav1.8-IR in which all 3 molars (M1, M2, M3) have retained wellstained odontoblasts in the pulp horns in the crown. A cross section of the incisor (INC) with strong Nav1.8-IR in all odontoblasts (OB) and gingival tissue (G, next to M3) are also shown. Nav1.8 is still missing from the roots. The continually erupting incisor (INC) had immature Nav patterns in its odontoblasts at all ages. (B) In year old rats, odontoblasts at the special tip dentine (*) of the pulphorn (PH) had weak Nav1.8-IR, it was missing at the furcation Zone VIII (F) and at reparative dentine Nav1.8-IR was lacking (RD). (C and D) In old rats Nav1.4 is still focused exactly beneath the molar special tip dentine (*) and at reparative sites in old teeth (RD). (E) For incisor odontoblasts the Nav1.4-IR remained extensive and strong in old rats, as did the ameloblasts and associated enamel-related structures (inset photo from the site indicated by arrow). Dots: Region of crown that attracts the most innervation. N: Nav. Scales: A, 0.5 mm; B–D, 0.1 mm. E: 0.25 mm.

Developing and ageing teeth

The shifting tissue structure of molars during development and ageing is shown in relation to key pulpal regions (Fig. 4). In newly-erupted immature molars, those changes are rapid for a few weeks during initial root formation when Hertwig’s root sheath is active. After root zones R1 and R2 have been formed in 7 week-old rats for first and second molars, the subsequent elongation of root pulp involves addition of cellular cementum around neurovascular channels that lack odontoblasts or dentine in zone R3. Pulp and dentine continue to change in adult teeth as dentine slowly thickens and the pulp gradually shrinks in response to molar use and crown attrition. The specialized tip region (tip, black) of rat molars (Figs. 2–4) and the pulp horns (PH, pink) were maintained during ageing despite extensive wearing down of the crown. The other regions became increasingly narrow, and the intercusps eventually completely filled in with dentine. Odontoblasts in the continuously erupting incisors had isoform-specific immature patterns for all Nav isoforms-IR at all ages (Figs. 3A,E and 6I–M). We analysed the intensity of odontoblast immunoreactivity for Nav1.1–1.9 in the eight molar regions (I–VIII) of pulp in all age groups (Table 1). The immature Nav patterns and their changes after maturation were different for each isoform. Some had much stronger odontoblast-IR in immature teeth than adults (Nav1.4, Nav1.7), some increased in mature crown odontoblasts but decreased in roots of mature teeth (Nav1.1, 1.2, 1.5, 1.8), one became increasingly intense during development in crown and root whilst avoiding the most innervated regions (Nav1.3), and two were expressed by dendritic cells (Nav1.6, Nav1.9) as well as by special odontoblasts.

3.4.

Gingival and periodontal patterns

Each isoform also had standard patterns of immunoreactivity in periodontal tissues (Fig. 6) that were present as soon as the teeth had erupted (gingiva: Table 1) and that served as positive controls for the immunohistochemistry. The molar pulp immunoreactivity for Navs differed from nearby periodontal ligament, alveolar bone, and gingiva (Figs. 2–4 and 6). We were especially interested in the immunoreactivity of the free gingiva and junctional epithelium where Nav1.1–1.9 had one of five patterns (Fig. 6): (1) the outer layers of free gingival epithelium were stained, but not inner layers or junctional epithelium; (2) basal layers of gingival epithelium and the junctional epithelium next to the teeth were stained; (3) labelling in basal and outer layers of gingiva and junctional epithelium; (4) no epithelial staining but positive reaction in subepithelial connective tissue, and (5) no Nav expression at all (Nav1.4). For each Nav isoform, the gingival pattern (or lack of it) was the same at all ages, and served as an intra-section positive control (Fig. 6A–F), as did nearby Nav-IR patterns for

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Fig. 4 – Rat molar development and ageing. Pulpal regions including the special tip (black) region are shown during root development in immature teeth of 3–4 weeks old rats, and in mature molars at 7 week, 3 months and 6–12 months. The 7 regions within molars have been colour coded, as indicated. The tip and pulp horn (PH) regions are retained in old rats, whilst the cervical crown (C), furcation (F) and root pulp (R1, R2) become much narrower. As teeth age, the roots are extended by production of cellular cementum at zone R3 whilst the width of the periodontal ligament between root and bone is maintained. HRS: Hertwig’s Root Sheath.

muscle (Fig. 6G), bone (Fig. 6H) and incisors (Fig. 6I–M). Nav isoform patterns for odontoblasts were not in synchrony with any of the gingival patterns.

3.5.

Rat incisors are continuously erupting teeth that turn over completely in about a month, and so their odontoblasts do not have a chance to become mature. Instead they are columnar and engaged in dentinogenesis at all ages, and their odontoblast patterns do not shift in older rats. We found that odontoblasts in incisors had similar intensities for the labial and lingual sides, with isoform-specific intensities (Fig. 3A and E). The exception was Nav1.3. It was intense in odontoblasts beneath the labial enamel-covered region (Fig. 6I), whilst remaining blank in other regions, and this was true for young and old rats. The other isoforms (e.g. Fig. 6J–M) had pale or moderate odontoblast immunoreactivity, along with distinctive patterns for ameloblasts and associated tissues. The associated ameloblasts for Nav1.4 had the most intense immunoreactivity of any structure in our sections (Fig. 3E).

3.6.

Fig. 5 – Intradental zones and Nav expression foci in mature adult molar. The most prominent Nav isoforms per zone in fully mature adults are underlined, and others that typify each region are shown in smaller font (as summarized in Table 1). The pulp horn in adult rats could be subdivided into tip region (I), dentine near the tip (II), well-innervated side of the pulp horn (III) and less innervated side (IV), as indicated. Cervical regions (V) had similar patterns for intercusp and side regions. Outward facing (interdental) sides of roots had distinctive patterns (VI), and then the furcation (VIII) and inward facing (interradicular) side of the roots (VII) had still further specializations. Odontoblast height varies amongst the different regions as indicated in magnified circles.

Incisor odontoblasts

Sodium channels in root pulp

Each Nav isoform had specific, predictable patterns in developing roots (Figs. 4 and 7; Table 1) that differed from one another, and in most cases differed from crown pulp. Examples show expression at the root tip in newly erupted teeth (Fig. 7A–E) that could be intense (Nav1.1), or included dense pulpal staining (Nav1.2), avoidance of the root apex pulp (Nav1.3) or expression in other pulp cells in addition to odontoblasts, such as fibroblasts (Nav1.2, Nav1.5) or dendritic cells (Nav1.9). In adults those patterns had changed at mature root apices (Fig. 7F–J). Additionally, we found that Nav1.4-IR and Nav1.8-IR were almost entirely absent from root odontoblasts (Fig. 3), and Nav1.6-IR was in pulpal dendritic cells.76 Root odontoblasts on the interdental side (Zone VI, Fig. 5) are compared at high magnification for Nav1.1, Nav1.3, Nav1.7 and Nav1.9 (Fig. 7K–N). Odontoblasts on the interradicular side

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Fig. 6 – Nav distribution patterns in gingiva and other tissues near rat molars. (A) Pattern-1 was found for Nav1.1, 1.6, 1.8, and 1.9 and labels the outer layers (arrow) of free gingiva strongly whilst the basal layers and junctional epithelium (JE)

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Fig. 7 – Sodium channel patterns in molar root pulp. Each pattern in developing or mature roots was highly predictable. This is shown for selected isoforms (A–E) during root elongation (3–4 week) as indicated and after closure of the root apex with onset of cellular cementum deposition (F–J) (7 week). Odontoblasts (OB) lining the growing tip of the root could be intense (A), weak (C) or unstained (D). Nav1.2 stained a plug of pulp cells at the root/periodontal junction (B and G). (K–M) High magnification views of R1 root pulp from old rats shows the odontoblasts (arrow) that face the interdental space (zone VI) for isoforms Nav1.1, Nav1.3, Nav1.7 and Nav1.9. Nav1.3 stains the odontoblast processes (OP) far into dentine, Nav1.7 only stains the process at the predentin (PD). Nav1.7 (M) at zone VIII (white arrow) had lost most odontoblasts in this old rat. N: Nav. Scales: 0.1 mm.

(Zone VII, Fig. 5) lacked Nav1.7 (Fig. 7M), but retained strong Nav1.3-IR (Fig. 7L). Nav1.9 was weak in root odontoblasts, but strong in dendritic cells that invaded the odontoblast layer (Fig. 7N). Nav1.3 was unusual in staining the entire odontoblast process of root zones VI–VII (7L), whilst Nav1.7 and 1.9 only extended into the process for the segment passing through predentin (7M, 7N). For all antigens, expression

ceased in root pulp where odontoblasts had completed radicular dentinogenesis (Fig. 7M).

3.7.

Sodium channels in crown pulp

We found that some of the channels (Nav1.1, Nav1.4, Nav1.7) were more widespread or more intense in odontoblasts of

were unlabelled. (B) Pattern-2 was found for Nav1.7 and had labelled inner layers (arrows) of free gingiva plus labelling of the junctional epithelium (JE). (C) Pattern-3 was found for Nav1.2 and 1.5, with labelled outer layer of gingiva plus staining of basal layers and JE. (D) Pattern-4 had no label in free gingival epithelium (EP), but there was connective tissue (CT) label, shown for Nav1.3. (E) Pattern-5 lacked any gingival staining and was found for Nav1.4. (F) Preadsorption (paa) control for Nav1.5. G. Muscle immuoreactivity (M) shown for Nav1.2. (H) Nav1.8 was found in osteocytes (OC) near non-specific labelling of bone marrow (BM). (I) Nav1.3 had focal intense immunoreactivity in the insicor odontoblasts on the labial side. (J–M) Every isoform has a specific pattern for incisor (INC) odontoblasts (shown here for Nav1.2, Nav1.5, Nav1.7 and Nav1.9) as well as for enamel associated tissues. The staining patterns in gingival and other tissues were present as soon as molars had erupted and so provided a standard positive level of immunoreactivity for all ages. Abbreviations: N: Nav; OB, odontoblast; Den, dentine; En, enamel; A, ameloblast; v, blood vessels. Scale bars: 0.1 mm.

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Fig. 8 – Sodium channels in molar crown odontoblasts. Nav1.1 (A–C) and Nav1.4 (D–F) were more intense in immature molars at 4 weeks (4W), increased at 7 weeks (7W), and then faded at maturation to moderate intensity for all odontoblasts of crown (Nav1.1) or to an intense residual focus at the special tip dentine (Nav1.4) (arrow, F) plus some labelled cells in cervical pulp (Zone V). Dots: Region of crown that attracts the most innervation. F: furcation, N: Nav. Scale bars: 0.1 mm. dots immature teeth and faded after maturation or became limited to focal areas (Figs. 8 and 9, Table 1). For example, Nav1.1-IR was strong in root and cervical odontoblasts of immature teeth, increased in crown odontoblasts by 7 weeks, and then

became weaker in adults (Fig. 8A–C). Nav1.4-IR was similarly intense in odontoblasts of roots and cervical crown at 4 weeks, then increased in pulp horns at 7 weeks, and then disappeared from most odontoblasts in adults, whilst remaining intense at

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Fig. 9 – Distributions of Nav1.7 in odontoblasts and nerves. (A–E) Nav1.7 was intense for all odontoblasts of immature teeth except where Nav1.7-IR nerve fibres were arriving (A–E). Boxes in A show locations for B–E. The Nav1.7 was more dense for the terminal web in some crown regions (B and D) than at the tip (C) or furcation (E). (F) By 7 weeks the Nav1.7 innervation was focused aymmetrically at the pulp horn odontoblast layer and associated dentine, but bypassed the tip region (*), whilst the odontoblast Nav1.7-IR had faded throughout the crown but was still well labelled in furcation and root pulp (F, arrows). (G) In older rats, the Nav1.7-IR innervaton appeared to combine features of CGRP-IR and PER-IR endings, both entering the dentine asymmetrically and making a dense plexus in pulp below the odontoblast layer. The Nav.1.7 patterns in older rats had a variety of patterns that could be similar to asymmetric CGRP (H and J) or to the more symmetric peripherin subpopulation of dental innervation (I and K). Odontoblast Nav1.7-IR is in intercusp odontoblasts but fades in pulp horn (K, white arrow), and some of the Nav1.7-IR nerve fibres form complex branched endings (K, black arrow) near the tip (*). Dots: Region of crown that attracts the most innervation. N: Nav. Scale bars: 0.1 mm.

the special tip region and in some odontoblasts of intercuspal regions (Figs. 2C, 3CD, and 8D–F). However, the incisor odontoblasts retained strong Nav1.4-IR at all ages (Fig. 3E). Odontoblasts expressed intense Nav1.7-IR in immature molars that down-regulated as soon as Nav1.7-IR nerve endings arrived (Fig. 9A–F), suggesting important odontoblast–nerve interactions during morphogenesis. The Nav1.7 neural patterns in adults were varied, either extending into dentine and forming the subodontoblast plexus (Fig. 9G and J) that matched CGRP asymmetric patterns (Figs. 2F and 9H), or they ended in the tip region in a more symmetrical arrange-

ment (Fig. 9K) as also seen for peripherin (Fig. 9I), suggesting the presence of Nav1.7 in more than one type of sensory ending. Odontoblasts retained weak to moderate Nav1.7-IR in some mature odontoblasts (Fig. 9G and K).

3.8.

Odontoblasts in innervated regions

There was considerable overlap for Nav1.8 and the dentinal innervation that typically contains many CGRP-IR nerve fibres (Figs. 10 and 11, Table 1). The nerve fibre endings were more widely distributed than the odontoblast Nav1.8-IR when

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intensity for Zone IV (Table 2). The tip region also had some Nav1.5-IR in immature and mature rats. There was also increased odontoblast-IR in the pulp horn for Nav1.1, Nav1.2 and Nav1.9 of adults compared to immature (4 weeks). Quantitative densitometry for Nav1.3, Nav1.5, and Nav1.8 in Zones III–IV showed similar asymmetry for the latter two that was opposite to Nav1.3 (Table 2).

3.9. Special odontoblast patterns for Nav 1.2, Nav1.6, Nav1.9

Fig. 10 – Double immunofluorescence for Nav1.8 and CGRP. (A and B) Nav1.8 (green, FITC) is found mostly for odontoblasts (OB) along the sides of the pulp horns in this young adult (2 months old) concentrating along the pulp/ dentine interface where the odontoblasts make a terminal web. CGRP (red) shows peptidergic nerve fibres that end in peripheral pulp and inner dentine of pulp horn and the intercusp region. CGRP nerve fibres had a more extensive distribution than the odontoblast Nav1.8. Nav1.8 was most dense along the side of the pulp horn (PH) that had the greatest innervation (arrow, white dot) but sparse at the pulp horn (PH) tip (*). N: Nav. Scale: 0.1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

detected by double immunofluorescence in young adults (Fig. 10). Locations of Nav1.3-IR and Nav1.8-IR in odontoblasts had inverse distributions (Figs. 2A,B and 11). In immature rats, Nav1.3 was intense throughout the molar root and much of the crown, including cervical and interradicular zones (V, VIII) (Fig. 2B) and became more intense in all regions during maturation (Figs. 2B, and 11A–C; Table 1). In old rats, Nav1.3-IR was increasingly found in the pulp horn (PH), though still missing, found in scattered odontoblasts, or reduced for zone III (Fig. 11D and Table 2). Odontoblast processes that extended into crown dentine were not stained by Nav1.3-IR (Fig. 11C and D), whilst those in root (Fig. 7L) and furcation (Fig. 11D) were stained by Nav1.3-IR in adults. Conversely, Nav1.8-IR occurred along the sides of the pulp horn where dentinal innervation was most dense (Fig. 11E–H), and was reduced in cervical regions and weak or absent from roots. Nav1.8-IR first appeared when dentine first began to innervate (Fig. 11E and F), it increased as crown innervation increased (Figs. 10 and 11G,H), it avoided tip regions (*) and reparative dentine (Figs. 3A,B, 10, 11H), and it had gradients of intensity similar to those for innervation.22,65 Nav1.5-IR was found in crown and root odontoblasts in immature rats, with a special asymmetric concentration for the newly innervating crown regions of immature teeth at four weeks (4W) (Fig. 11I and J), and with greater intensity for the more innervated pulp horn regions (zone III) in mature molars (Fig. 11L–N) compared to less

Nav1.2-IR was seen in odontoblasts on either side of the special tip zone of newly mature and young adult molars (Fig. 12A–C), whilst it was also expressed at lower intensities in other regions (Table 1). Nav1.6-IR was widespread in immature odontoblasts, and then concentrated in odontoblasts at intercusp or sides of cervical Zone-V of adults (Fig. 12D and E). Nav1.9-IR was weak in young teeth (Fig. 12F) and gained intensity in adult molars (Fig. 12G–I), especially in scattered intensely-stained cells within the odontoblast layer (Fig. 12H and I). Those scattered odontoblasts with Nav1.9-IR show that there can be varied phenotypes within some zones. Nav1.9-IR was also found in large pulpal dendritic cells that often extended thin processes into the odontoblast layer (Fig. 7N and 12F–I).

3.10.

Patterns for TRPA1 and Nestin in rat molars

We found that the focal immunoreactivity of Nav1.6 at odontoblasts of intercusp and cervical crown (Fig. 12D and E) was similar to the location of the pain-related transient receptor potential TRPA1-IR in newly mature (7 week) and adult rats (Fig. 13A–C). TRPA1 also had a temporary expression at the growing tip of the roots (Hertwig’s root sheath) of immature molars (Fig. 13A). Nav1.6-IR (Fig. 12C–E) and TRPA1IR were also found for osteoblasts along alveolar crest bone (Figs. 12C and 13A–C). A very different distribution was found for the odontoblast marker nestin (Fig. 13D–G) that was present in all pulpal regions of the immature, mature and ageing rat molars including the interradicular regions that lose some Nav isoforms during ageing (Fig. 7M). Nestin-IR was also in periapical cellular cementum but otherwise sparse in periodontium.

4.

Discussion

4.1.

Multiple odontoblast phenotypes

The Nav patterns in rat molars showed that odontoblasts have a variety of phenotypes in mature teeth, and that the special crown regions are maintained from their initial maturation at root closure until at least one year (Fig. 4). This work expands the concept of successive transcriptomes during odontoblast morphogenesis and maturation6 by showing that mature odontoblasts specialize locally to have different expression of voltage-gated sodium channels for their specific region of the tooth. Each Nav isoform-IR had unique patterns, and immature teeth differed from adult. We propose that there are at least eight phenotypes for odontoblast expression of Nav

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Fig. 11 – Sodium channels related to crown innervation. (A–C) Nav1.3 was initially missing from pulp horn innervation zones (A) and that continued in young adults (B and C), whilst being expressed intensely in other regions (Zones V–VIII). It also extended into the odontoblast processes in root dentine but not crown. There was a variety of Nav1.3-IR intensities in the odontoblast layer of adult molars (C, inset). (D) Old adults increasingly gained Nav1.3 in the pulp horn (PH) (black arrows) including near reparative dentine (RD), but still lacked it in most cells of zones III–IV (white arrowheads). (E and F) Nav1.8 was found to increase in young 4 week old (4W) rats exactly where initial innervation of dentine was found on anterior side of maxillary (mx) molar pulp horns (E, arrows, dot) and posterior side of mandibular molar pulp horns (md) (F, arrows, dot), where it was concentrated at the pulp/dentine interface. (G and H) In mature rats the Nav1.8 distribution matched innervation territories of zones III–IV but skipped the tip areas (*) and was less intense in cervical zone V. (I and J) Nav1.5 was also found oriented towards newly innervating dentine (arrows, dots) in 4 week old rats with opposite orientation for maxillary and mandibular pulp horns, occurring in the arriving nerve fibres as well as the associated odontoblasts. (K and L) Nav1.5 increased in odontoblasts throughout the tooth by 7 weeks (7W), and was found in most odontoblasts of mature rat crown zone III (black arrows), including the tip zone (*), with weaker staining for zone IV (white arrowhead). (M) Preadsorption of the primary antibody (M, paa N1.5) removed all staining shown here for the pulp horn (PH). (N) Nav1.5-IR was found in the odontoblast (OB) cell bodies and in cells of the pulpal cell rich zone (inset, CFZ). N: Nav. Dots: Region of crown that attracts the most innervation. Scales: 0.1 mm.

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Fig. 12 – Focal expression of Nav isoforms. (A–C) Isoform Nav1.2-IR was initially found in all developing odontoblasts, and became more intense on either side of the pulp horn tip (zone II) and near the root apex (see Fig. 7B and G). (D and E) Nav1.6IR was concentrated in zone V of cervical crown and occurred weakly elsewhere. (F) Nav1.9-IR in 4 week old rats was found in developing odontoblasts of crown except for tip (*) and in dendritic cells scattered in the pulp. (G and H) By 3 months (3M) the Nav1.9-IR was weak or moderate in most areas, still bypassed the tip, and also was found at strong intensity in scattered odontoblasts of crown (H, small arrows; I, grazing section of zone V). Dots: Region of crown that attracts the most innervation. N: Nav. Scales: 0.1 mm.

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Fig. 13 – Transient receptor potential A1 compared with nestin. (A–C) TRPA1 receptor-IR had similar patterns to Nav1.6 in young rats (see Fig. 12C–E), occurring most intensely in cervical odontoblasts (Zone V) in 7 week old (7W) and 3 month (3M) rats (A and B insets show intensity change at border between pulp horn and cervical crown). TRPA1-IR also focused in the cells of Hertwig’s root sheath (HRS) at the growing root apex at 4 weeks (A) and occurred in osteoblasts (A–C). (D–G) Nestin-IR was a strong marker for all odontoblasts of immature rats at 1–3 weeks (1–3W), young adult (3M) and old rat molars at one year (1 year), including those of tip regions. In the adult rats, odontoblast processes also had nestin-IR, especially in crown regions II–V and root zone VI. N: Nav. Scales: 0.1 mm Dots: Region of crown that attracts the most innervation.

isoforms in mature rat molars (Fig. 5), and that those suggest different odontoblast functions at (I) the pulp horn tip, (II) immediately adjacent to the tip, (III) the pulp horn side with dense innervation, (IV) the pulp horn side with less dense

innervation, (V) the cervical crown (intercusp and side regions), (VI) the root odontoblasts that face ‘out’ at R1 and R2 towards other teeth, (VII) the interradicular side of the root at zones R1, R2, and (VIII) the furcation odontoblasts.

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Only Nav1.8-IR and Nav1.5-IR overlapped the dentinal innervation locations and gradients, although reduction of Nav1.3-IR in those same innervated regions may also contribute to neural locations or functions (Tables 1 and 2), and Nav1.1 was expressed at moderate level throughout the mature crown and for Zone VI. Nav1.5 and Nav1.8 are both tetrodotoxin resistant and may be contributing to anaesthesia resistance in inflamed teeth44,67 in some as yet undefined odontoblast:neural interactions. Interestingly the pattern for odontoblast TRP-A1 shared similarities with Nav1.6-IR, and TRPA1 has been linked to a variety of sensory transduction mechanisms some of which affect pain.22,80,82 Many other molecules such dentine specific phosphoprotein (DSPP) and nestin (Fig. 13)83,84 occur in all odontoblasts, whilst others are focused in crown (dentine matrix protein-1, DMP-1)6 or root odontoblasts of mature teeth (protein gene-product, PGP9.5).85 Similarly different isoforms of a molecular family, such as connexins 26, 32, and 43 are expressed at different developmental stages or by functional sub-types of odontoblast.36 Our demonstration of eight odontoblast phenotypes for Nav expression is the first time that such complex diversity has been demonstrated for mature odontoblasts for the isoforms of a single molecular family, and the scattered immunoreactivity for Nav1.9 and Nav1.3 in crown odontoblasts suggests that more phenotypes will be found. Exactly how these channels regulate the functions of different sets of odontoblasts remains to be determined, and may include adhesion activities that have been shown for the beta subunits of sodium channels.86

4.2.

Voltage-gated sodium channels

Even though the nine mammalian isoforms for the alpha subunit of voltage-gated sodium channels are very similar in sequence, they differ in their electrophysiological properties, drug sensitivities, and cellular/intracellular locations.44,45,73– 75,87,88 Initially, Nav1.1, Nav1.2, Nav1.3 and Nav1.6 were mainly observed in the central nervous system, and Nav1.7, Nav1.8 and Nav1.9 were predominantly localized in the peripheral nervous system. In contrast, Nav1.4 and Nav1.5 were identified as the main skeletal muscle and cardiac muscle sodium channels, respectively.87 Whilst this trend holds, recent studies now suggest that many tissues such as cardiac tissue89,90 express multiple voltage-gated sodium channels in addition to the main isoforms originally identified in these tissues, and all nine Nav isoforms have been found for different stages of sperm morphogenesis.91 Here we have identified the expression of all nine voltage-gated sodium channels in developing and mature tooth odontoblasts of rat molars, with nearby periodontal tissues also having characteristic patterns for all the Nav isoforms except Nav1.4. Electrophysiological studies originally showed that voltagegated TTX-sensitive sodium currents in cultured human pulp cells are capable of generating action potentials.11,12,19,47–49 However, the purpose of that sophisticated signalling remains to be determined, and may enable local responses to mechanical loading that lead to appropriate matrix production. Studies of peripheral nerves in both humans and animals suggest that several voltage-gated sodium channels are

pivotal in pain transmission. These channels include Nav1.7, Nav1.8 and Nav1.9 that are upregulated in painful human teeth,61,66,67 and Nav1.3, that is upregulated after nervous system injury.92 Notably, these channels, along with Nav1.5, are the Nav isoforms that increased or remained at high levels in odontoblasts after maturation of the tooth, raising the possibility that they may play an important role in mature tooth sensitivity. Nav1.3 is thought to be the major channel in embryonic central and sensory neurons that is subsequently reduced in neonates, absent in adult dorsal root ganglia (DRG), and expressed at low levels of expression in some adult human tissues.92–95 However, in teeth the Nav1.3IR patterns intensified as odontoblasts matured in rat molars, except for odontoblasts in the most innervated regions of crown. Relatively high levels of Nav1.3 channels are present in adult sympathetic ganglion neurons, and they are upregulated in dorsal root ganglia,92–95 dorsal horn96 and thalamic97 neurons after peripheral nerve injury92,98 Nav1.7 is expressed in sensory, sympathetic and myenteric neurons, it increases in nerves of painful human teeth,66 and has been linked genetically to pain disorders in humans.99 Nav1.9 is expressed in sensory and sympathetic neurons whilst Nav1.8 is specific to sensory neurons and is localized mainly in DRG and trigeminal ganglion100 but has also been observed in peripheral axons, free nerve terminals in teeth101 and in skin and cornea.102,103 Multiple inflammatory mediators have been shown to differentially regulate Nav1.8 current in neurons,104–107 suggesting an important role in inflammatory pain. Nav1.9 has been shown to increase in pulp nerves of symptomatic (painful) teeth,61 whilst Nav1.8 protein increased in homogenized pulp from painful human teeth.67 Our data now indicate that western blot analyses of Navs in homogenized tooth pulp may have included proteins from odontoblasts and deeper pulp as well as from nerve fibres.

4.3.

Sodium channels in non-neuronal cells

Here we have shown immunoreactivity for voltage-gated sodium channels (VGSC) in odontoblasts, the cell rich pulp region, gingiva, periodontal ligament fibroblasts, alveolar bone and dendritic cells, along with previous demonstration of Nav1.6-IR in alveolar osteoclasts, odontoblasts, pulpal macrophages and dendritic cells.51 As noted above, Nav isoforms are essential for cardiac muscle, skeletal muscle and neuroglia, related to their specialized excitation–contraction mechanisms and neural interactions. VGSC have also been found in a variety of other non-neural cells [reviews: 108,109], such as cardiac and sinoatrial nodal fibroblasts,110,111 T-lymphocytes and macrophages,112,113 endothelial cells,114 neuroendocrine cells,115 and prostate or breast cancer cells where tetrodotoxin can reduce metastatic behaviour.116,117 Osteoblasts and odontoblasts share many similarities including ion channels and mechanosensitivity related to ability to sense and adapt to mechanical strain.19,52,118,119 VGSC are the only ion channels whose beta subunit(s) also enable other cell functions such as cell:cell adhesion, membrane stabilization via ankyrin and other proteins, and regulation of trafficking dynamics,74,85,108 all of which may be important for odontoblasts. In many cases (neural and non-neural) there is

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posttranslational splicing that alters the function of the sodium channel,109 but in other cases action potentials were found in non-neuronal cells,110,112 as has also been found for immature odontoblasts in vitro.12,19,49 The mechanosensitive network of fibroblasts in heart has especially interesting similarities to the pulpal fibroblast and odontoblast networks, because they are linked by many gap junctions, they are mechanosensitive, and they affect cardiomyocytes via paracrine mechanisms.108,109,120 It should be noted that both cardiac and pulpal fibroblasts as well as odontoblasts express Nav1.5-IR (Fig. 11).120 There are also similarities between dental mechanoreceptors and the vibro- and mechano-sensitive lateral line and organ of Corti systems, in which support cells are linked by gap junctions amongst themselves but not with the neural sensory receptors,121 just as odontoblasts and pulpal fibroblasts are linked together by gap junctions but not with the nearby nerves.35–37

4.4.

Nerve–odontoblast interactions

Odontoblasts must be amongst the most complex cells of the body, considering that they make and maintain dentine matrix and regulate its calcification, as well as having epithelioid functions including making a terminal web,27–35 immune and antimicrobial functions,39–43 and responsibility for making and regulating dentinal fluid and dentine permeability.16,17 In addition they have mechanosensivity,12,19– 21,46,47 as do many other non-neuronal cells, noted above, and they move in response to dentinal fluid movements that follow painful stimuli to teeth.24,25,62–65 Our results show that immature patterns differ from mature, and thus a full understanding of odontoblast functions will require more work with fully mature teeth. The extent to which mature odontoblasts affect dental innervation could involve (a) direct actions by odontoblasts that influence dental pain and/or mechanosensitivity, or (b) indirect paracrine actions that can sensitize or modulate neural functions. Nerve–odontoblast interactions also include extensive effects of sensory nerve fibres on their target tissue that would affect odontoblasts and other pulp cells via release agents such as glutamate and calcitonin gene-related peptide. These powerful neural efferent actions include regulation of fibroblast gap junctions122, local cells, vasculature, immune cells, and dentinogenesis.69,123–128 Direct actions of odontoblasts on dental innervation have been proposed based on the ion channels and excitability of odontoblasts and the proximity of some nerve endings near the odontoblast terminal web, gap junctions, cilium and dentinal process.12,18–21,27–31,46–49,129 It is highly likely that there are Nav-dependent functional differences amongst each of the eight mature odontoblast phenotypes, and it may be that one or more of those subtypes has direct effects on nearby nerve fibres, whilst other phenotypes act locally to maintain pulp and dentine. Pain studies in human volunteers show the importance of fluid shifts in dentinal tubules (hydrodynamic mechanisms) that also cause odontoblasts to move24,25,62–65 when tooth stimulations hurt, or in animal studies.26 It may be that nerve fibres are highly sensitive to movements of their tissue framework (odontoblasts), as

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suggested by Gunji.29 However, it has not yet been proven that odontoblasts directly affect neural activity, and nerve fibres possess sufficient membrane receptors to enable their independent responses to that fluid movement,54–59 in some cases at such rapid rates that odontoblasts cannot be directly involved.54 Indirect actions have been proposed based on the demonstration that some of the nerve fibres in teeth are purinergic and can respond to release of ATP by odontoblasts or other pulp cells.60,130 Furthermore, the expression of two pain-modulating endothelin receptors (ETA and ETB) in the odontoblast layer of developing and mature rat teeth131 shows that odontoblasts can respond to endothelin and thereby may participate in modulating neural activity, as do keratinocytes in skin132,133 and other integument epithelia134 following endothelin stimulation. That possibility fits with demonstrations that neural activation in teeth is greatly affected by pulpal conditions including inflammation.69,70 Finally, our results are also consistent with the idea that non-neural tissues express ‘neural’ molecules simply for local sensory-response functions without affecting neural activity.

4.5.

Crown/root differences

Crown development can be successful in many different environments so long as the stem cells of the cervical loop are active and still regulated by Fgf10 and Notch1.135 For example, when tooth buds are transplanted to the cheek or to striated muscle or in vitro, they grow into fully formed crowns, but subsequent root growth can be problematic.136 Here, we have shown striking crown/root differences in odontoblast expression of Nav isoforms that may play important roles in tooth morphogenesis in situ, and may underlie some of the problems for root regeneration. We were especially interested that the odontoblast process was fully stained for Nav1.3 in root and furcation odontoblasts, but not for crown (Figs. 7L and 9B), showing a very different intracellular distribution when dentine is covered by enamel compared to dentine covered by cementum. Many other studies have also found differences in expression in crown and root6,12,32,82,137–139 including definition of root specific stem cell signals,135 just as occurred for some of the Nav isoforms reported here. In addition, the odontoblasts associated with the furcation dentine (Zone VIII) and with the interradicular facing dentine (Zone VII) of the roots differed from crown and from interdental-facing root odontoblasts (Zone VI) for most Nav isoforms. Similar differences have been found for expression of heat shock protein-25.140

5.

Conclusions

Our description of the expression of immunoreactivity for nine Nav isoforms in rat teeth shows that mature molar patterns differed from immature teeth and from patterns in nearby gingiva, periodontal ligament, alveolar bone and continually developing incisor teeth. Odontoblasts were the key cells expressing Nav isoforms in rat teeth, but the cell rich zone (fibroblasts, stem cells, neuroglia), nerve fibres and

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dendritic cells in pulp also expressed some of the Nav isoforms. Each of the nine Nav isoforms had a different spatial/temporal set of patterns in odontoblasts that collectively defined at least eight different mature odontoblast phenotypes (Fig. 5). Further analysis of odontoblast phenotypes will help understand how these cells orchestrate many aspects of odontoblast function that appear to be specialized for different tooth regions. The tetrodotoxin-resistant Nav1.5 and Nav1.8 were prominently expressed by odontoblasts in innervated regions of the crown as well as by the cell-rich zone (Fig. 11). Nav1.8 most closely overlapped regions of innervated dentine, and it was concentrated in the terminal web region of the pulp horn during early stages of dentinal innervation (Figs. 10 and 11). Whether that overlap indicates specific nerve–odontoblast junctions and interactions that influence dental pain mechanisms and perceptions, and/or whether it has other purposes, remains to be determined. It is certainly possible that one or more of the phenotypic regions within mature rat molars includes specializations to link odontoblast activity to nerve activity. However, the odontoblasts may also have ‘borrowed’ neural genes to enable their detection of tooth use, damage or infection, in order to better adjust their dentinogenesis, immune functions and pulp–dentine barrier requirements. The present study of expression of all nine Nav isoforms in rat teeth reveals the most complex variation in mature odontoblast phenotype that has yet been defined.

Funding Partial funding by NIH grants DE05159 (MRB) and DE13531 (REW), by Amer. Assoc of Endodontists Research Foundation (MRB), and by local research funds in University of Washington Department of Anesthesiology and Pain Medicine (MRB), and by UW Dental School Research Fund (MRB).

Competing interests None declared.

Ethical approval Ethical approval by University of Washington Institutional Animal Care and Use Committee, protocol #2074-01.

Acknowledgments We thank Dr. Orapin V. Horst for helpful comments on the manuscript. We gratefully acknowledge the helpful image processing and figure construction by Paul R. Schwartz. We also thank Dr. Orapin Veerayutthwilai Horst for helping with the initial pilot project, and Ian P. Townsend, Norma L. Anderson, Rosa M. Crumpton, Nadine A. Luis, and Thuy Vien for expert sample preparation. The authors have no conflicts of interest for this work.

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