Okajimas Folia Anat. Jpn., 74(6): 317-328, March, 1998

A Histological Study of the Organic Elements in the Human Enamel focusing on the Extent of the Odontoblast Process

By

Yuuki OGITA, Yasutomo IWAI-LIAO and Yoshikage HIGASHI

Department of Oral Anatomy, Osaka Dental University, 8-1, Kuzuhahanazono-chuo, Hirakata-shi, Osaka 573-1121, Japan

- Received for Publication , February 12,1998-

Key Words: Histology, Organic elements, Human enamel

Summary: Topographic and tomographic studies were conducted on the organic elements occluded in the enamel of premolars removed from young orthodontic patients by using light (transmitted) microscopy, confocal scanning laser microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) on ultrathin sec- tions and freeze-etching replicas, and energy dispersive spectroscopy (EDS) X-ray microscope (EDX) analysis. The present fine structure study aimed in particular to determine the fine structure of the enamel spindle and the extent of the odontoblast process. Organic elements in the ground-sectioned enamel corresponding to simple projections and enamel rods/spindles, enamel tufts and lamellae were identified by conventional light microscopy and subsequently examined by CLSM. Both light microscopy and CLSM indicated that a number of enamel spindles were measured about 50 μm in length, some 4-7 μm in thickness and were mostly confined to the cuspal summits and conformed to previous descriptions. SEM examination revealed some simple projections extending from the dentine into the enamel as well as clearly identifiable enamel spindles; the enamel spindles were structures intervening enamel prisms and showing morphological complexity by branching and convergence of the distal endings of the invading organic structure from dentinal tubules. EDX- analysis revealed that enamel tufts, lamellae, and spindles contained less phosphorus and calcium elements than enamel prisms. The enamel spindles had a higher content than tufts or lamellae, but this may be the result of contamination from surrounding enamel. Both conventional ultrathin-section and freeze-etching replica TEM evaluation of the dentino-enamel boundaries in particular suggested that simple projections and enamel rods/spindles were extensions of the odontoblast processes trapped in the enamel during early amelogenesis. In contrast, both SEM and TEM obser- vations failed to identify dentinal tubule, peritubular (intratubular) dentine, membranous structures or lamina limitans surrounding the enamel spindles and simple projections ocduded in the human enamel.

It is generally agreed that simple projections, tubular (intratubular) dentine of dentinal tubules enamel rods and spindles are odontoblast processes (Garant, 1972; Holland, 1976; Thomas, 1983; (OPs) that occasionally invade the enamel (Tomes, Thomas and Carella, 1983; Thomas and Payne, 1856;Fujita and Yuasa, 1949;Hodson, 1952;Avery, 1983; Thomas, 1984; Thomas and Carella, 1984; 1986; Mizuhashi and Suga, 1990). Despite the de- Weber and Zaki, 1986;Torneck, 1994). Conversely, velopment of various preparation techniques for other investigators, also using SEM, TEM and stabilisation and minimisation of artefactual dam- immunocytochemical methods, have demonstrated ages on OPs, however, the extent of the OP within that OPs in dentinal tubules frequently ran the dentinal tubule remains a matter of debate throughout the full thicknes of the dentine from the (Tomeck, 1994). Some transmission electron micro- surface of dental pulp to the DEJ in vivo (Sigal et scopy (TEM) and scanning electron microscopy aL, 19841; Sigal et aL, 19842; La Fleche, Frank and (SEM) studies have not only described that the OP Steuer, 1985; Sigal, Aubin and Ten Cate, 1985; did not extend beyond the inner half of dentine, but Torneck, 1994). also pointed out that the structures in the outer- Recently, confocal scanning microscopy, in par- third of coronal dentinal tubules, extending to the ticular confocal laser scanning microscopy (CLSM) dentino-enamel junction (DEJ), were not OPs, but connected to an image analysis system was applied rather that they were extracellular matrix and an to non-destructive specimens for tomographic and organic sheet-like lamina limitans lining the peri- three-dimensional study of hard tissues (Boyde

317 318 Y. Ogita et aL and Martin, 1984; Carlsson et al., 1985; Howard et prepared according to the freeze etching techniques al., 1985; Boyde, 1986; Benn and Watosson, 1989; using a Hitachi HFZ-1 freeze etching apparatus Nakamura and Kawata, 1990; Kodaka, Debari and (-115°C, 1 x 10-5 Torr; Hitachi, Tokyo, Japan). Yamade, 1991; Ohya et al., Tanaka and Nishikawa, After standard platinum-carbon replicas were pre- 1995). In the present study, the relationships be- pared, biological materials were digested with tween the organic elements included in the human bleach, and then the replicas were rinsed and enamel and the odontoblast process extending in mounted on grids for TEM study (Richard, Richard the dentine were initially examined using CLSM. and Tung, 1991). Then, the structures presumed to be enamel rods/ To view organic elements in the enamel, some spindles and simple projections were analysed and premolars were not demineralised but instead were compared with the surrounding enamel prisms. For either longitudinally or laterally ground-sectioned further three-dimensional study of the enamel rod/ after prefixing with the same aldehyde fixation spindle and simple projections occluded between solution. The samples were etched with 0.1 N hydro- enamel prisms, HC1-etched ground-sectioned and chloric acid (HCI; for about 10 seconds), stained ethylenediamine tetraacetic acid (EDTA)-demin- with 0.27% Ziehl's carbolfuchsin for localisation eralised specimens were investigated using conven- of poorly calcified elements in the enamel. The tional SEM. Subsequently, conventional TEM of specimens were then dehydrated, dried, mounted, EDTA-demineralised specimens and freeze-etching examined and the tomographic images were ana- replica TEM, were conducted on the fine structure lysed at 0.5-2 pm intervals using an Olympus GB- of OPs in the dentine and enamel rods/spindles and 200 CLSM (argon ion laser, excitation wave length: simple projections at the DEJ. 488 nm; using CH1: 0-515 and BP-535, CH2: 0-570 and 0-590 filters; Olympus, Tokyo, Japan) con- nected to an image analysis system (CLSM-GB 200 Materials and Methods application ver. 2.13; Compaq Prolinea-4100 com- puter, USA), and photographed using an Avio FR- Non-carious premolars were obtained from 3000 film recorder (Avionics, Tokyo, Japan) by orthodontic patients, aged 10 to 20 years, under modifying the methods proposed for studying tooth electro-acupuncture analgesia to avoid adverse decay (Tanaka and Nishikawa, 1995). Additionally, effects associated with anaesthetics (Ueki, 1992). some specimens from undemineralised samples The teeth were dipped in chilled 0.1 M sodium were also ground, coated either with gold in the (Na)-cacodylate buffer solution, split into halves Eiko IB-3 ion coater or with carbon in a Hitachi with a diamond disk mounted in a dentist's hand HUS-4GB evaporator, and prepared for SEM and piece and immersed immediately in chilled alde- EDS X-ray microprobe analysis. EDS X-ray micro- hyde fixatives (2.5% paraformaldehyde + 2.0% probe analysis was conducted using a Horiba Super glutaraldehyde; pH 7.2; buffered with 0.1 M Na- Xerophy detector (set at inclination angle of 35.0°, cacodylate; 0-4°C; 24-48 h). The samples were 10.0 kV accelerating voltage, 2 x 10-10 amperes then subjected to EDTA (ethylenediamine tetra- probe current and a live time of 100 seconds; acetic acid) demineralisation (2.5%; buffered with Horiba, Kyoto, Japan) connected to a Hitachi S- 0.1 M Na-cacodylate; 0-4°C) for six to eight 2700 SEM and a Horiba EMAX-5770X X-ray mi- months. Upon completion of decalcification, the croanalyser using the ZAF (atomic number effect, samples were cut into small blocks, rinsed in several absorption effect and fluorescence excitation) com- changes of Na-cacodylate buffer and postfixed with pensation method. In the present study, both pure osmiun tetroxide (1 % 0504; buffered with 0.1 M gallium phosphide (GaP) and calcium carbonate Na-cacodylate; 0-4° C; 4-8 h). Dehydration was (CaCO3; C.M. Taylor Corp., Stanford, Calif., USA) crried out through a graded series of cold ethyl were used as standard specimens. alcohol. Some of the demineralised samples were embedded in epon 812, ultrathin-sectioned, stained, observed and photographed under a Hitachi HU- Results 7100 TEM (Hitachi, Tokyo, Japan) following con- ventional methods. Other demineralised samples Organic elements in the enamel corresponding were dried in a Hitachi HCP-1 critical point drier, to simple projections, enamel rods/spindles, enamel ion-coated with gold in an Eiko IB-3 ion coater tufts and lamellae deeply stained with carbolfuchsin (Eiko Engineering, Mito, Japan), mounted on were identified in HC1-etched ground specimens stubs, examined and photographed under a Hitachi using light microscopy and then examined using H-4100 field-emission SEM (Hitachi, Tokyo, Japan). an Olympus GB-200 confocal laser scanning micro- Some non-demineralised sectioned samples were scope (CLSM). Enamel spindles showing an en- The Extent of the Odontoblast Process 319 larged ending deeply invading the enamel were them (Figs. 3 & 4). However, the present CLSM measured as about 50 gm in length and 4-7 gm and SEM study could not define exactly whether thick; and they were mostly found in the cuspal these were invaded odontoblast processes (OPs) summits (Figs. 1 & 2). By image processing, accu- or other occluded organic elements. Therefore we mulation of tomograms of the ground section sug- conducted further transmission electron microscopy gested that simple projections and enamel rods/ (TEM) on enamel rods/spindles and simple projec- spindles occluded in the enamel were continuous to tions, in particular on those distributed at the DEJ. the contents in dentinal tubules (Fig. 2). Scanning The present conventional TEM of the DEJ in electron microscopy (SEM) on the dentino-enamel epon-embedded ultrathin sections clearly demon- junction (DEJ) of HCI-etched ground-sections also strated many OPs in the dentinal tubules extending demonstrated many simple projections and enamel into the enamel and the cytoplasm of OPs con- rods/spindles in the enamel; these were structures tained some electron-dense organelles and micro- extended from the contents of the dentinal tubules tubules (Figs. 8a-9b). The periodontoblastic space of the underlying dentine (Figs. 3 & 4). Subse- between the OP and matricial collagen fibres of quently, EDS analysis was conducted on the struc- the demineralised peritubular dentine, was found to tures supposed to be organic elements occluded in contain some collagen fibres and an electron-dense the enamel. The results indicated that the struc- distinct electron-dense membranous structure mor- tures contained less weight percent (wt %) of both phologically similar to the lamina limitans lining calcium (Ca) and phosphorus (P) elements than the either the cell membrane of OPs or the tubular wall surrounding enamel prisms, whereas the thin enamel (Figs. 8a & 8b). Serial ultrathin sections enabled spindle/rod and simple projection showed higher us to trace the OPs in the dentine revealing that Ca and P content than the enamel tuft and lamella some of their distal endings were occluded in the (Table 1). demineralised enamel (Figs. 9a & 9b). SEM of EDTA-demineralised samples clearly By using freeze-etching techniques, TEM obser- revealed that many projections, some of which vation of the replicas showed dentinal tubules con- had an enlarged ending, which were presumed to taining OPs that branched and intruded into DEJ be enamel rods/spindles and simple projections, (Fig. 10a). Some OPs with lateral branches were extended from the dentinal tubules (Figs. 5 & 6). freeze-fractured at the cell membrane to show the These structures were included in the enamel at a protoplasmic face (PF) and exoplasmic face (EP) characteristic oblique angulation (Fig. 6). The mor- byTEM observation (Fig. lob).The images sug- phological complexity of the enamel spindle, show- gested that most of the peripheral dentinal tubules ing branching and convergence of the endings was at the DEJ were actually filled with OPs, and that shown in the present SEM (Fig. 7). Additionally, also there was no periodontoblastic space evident to SEM of the HC1-etched specimens indicated that contain the large number of intratubular mem- the enamel rod/spindle and simple projections were branous structures in the dentinal tubules. embedded directly into surrounding enamel prisms; no dentinal tubules or hypermineralised peritubular (intratubular) dentine were observed surrounding

Table 1. Weight percents of P and Ca in the enamel spindle, tuft, lamella and their surrounding enamel prism using EDS X-ray microprobe analysis 320 Y. Ogita et aL

Discussion had higher levels than the enamel lamina and tuft. This may be explained by the fact that the inclina- A previous transmission electron microscopic tion angle used in the present EDS analysis on the (TEM) study observed that, even in germ-free rat thin enamel spindle would result in a characteristic dentine, the odontoblastic process (OPs) may not X-ray pattern that was generatedfrom the sur- persist as a viable cytoplasmic extension all the way rounding highly calcified enamel prisms. out to the dentine-enamel junction (DEJ). How- Our scanning electron microscopy (SEM) on the ever, many dentinal tubules, in particular at the DEJ revealed many simple and enlarged projec- distal portions, appeared either empty or occluded tions that extended from dentinal tubules into the by the electron-dense lamina limitans composed of clefts of the enamel. The morphological complexity collagen and amorphous material. The same study due to division, union and bulge of the enamel therefore suggested that dentinal tubules were not spindle endings was evident (Avery, 1986). These completely occupied by OPs but that a normal re- were thought to be substances included in the cession of the processes occurred with or without a enamel at the characteristic oblique angulation, deposition of occluding substances (Garant, 1972). which formed between ameloblasts and enamel Other TEM studies on the cat and human dentine prisms during amelogenesis (Sharawy and Yaeger, have also shown that OPs were limited to the inner- 1986). The dentinal tubule and peritubular (intra- third to inner-half of the dentine. These results tubular) dentine were not observed between the were observed irrespective of either variations in occluded enamel spindles and enamel prisms. composition, concentration and osmotic pressure Using light microscopy, CLSM and SEM, we of aldehyde fixatives, or duration of fixation and observed that enamel spindles were morphologi- the method of tooth preparation (BrannstrOm and cally very similar to the distal endings of OPs, Garberoglio, 1972; Holland, 1976; Thomas, 1983). which have been included in the enamel. However, However, one study noticed that dentinal tubules in we could not rule out the possibility that they only the radicular dentine of the cat developing apex did represented the surface or the accumulation of contain OPs (Holland, 1976). organic elements that mimic an OP (Avery, 1986; Contrasting ultrastructure studies on OPs have Torneck, 1994). A TEM study pointed out that a claimed that they extended throughout the entire structure similar to an OP was actually an electron- thickness of the dentine from the pulp to the DEJ dense structure limiting the peritubular (intra- in mice and monkeys (Corpron and Avery, 1973; tubular) matrix and lining the inner aspect of all Kelly, Bergenholtz and Cox, 1981). Further, using dentinal tubules. This structure, the lamina limi- labelled antibodies against intracellular proteins, tans, was clearly distinguishable from the cell some researchers have also shown that the majority membrane of the odontoblast process. The same of dentinal tubules contained cellular components study also reported that the amount of peritubular along their entire extent (Sigal et al., 1984'; Sigal matrix was considerably increased over that seen et aL, 19842; Sigal, Aubin and Ten Cate, 1985; in the inner-third and was present in all tubules Torneck, 1994). Other TEM studies have observed examined (Thomas, 1983). Further, some SEM and that the elongation of cytoplasmic processes of TEM studies have stated that the organic perio- rat odontoblasts was coincidental with continuous dontoblastic space, for the elaboration of the peri- deposition of dentine; therefore it was reasonable tubular dentine, is built up by uncalcified collagen to consider that OPs would traverse the whole fibrils and an amorphous substance lining the thickness of the dentine. Dentinal tubules contain- peritubular matrix (Bradford, 1950; Frank, 1966; ing shortened OPs were proposed to be sites that Isokawa et aL, 1972; Thomas, 1983). had narrowed and become occluded with mineral Some studies have suggested that conventional deposit (Garant, 1972; Avery, 1986). dentine preparatory techniques for TEM which The present tomographic study using confocal failed to demonstrate OPs in periopheral dentine laser scanning microscopy (CLSM) demonstrated were due to contraction of the OP; therefore cryo- that the enamel spindle, measuring about 50 gm in fixation was suggested to be a useful method for length and 4-7 gm in thickness, was included in the preventing artefacts when demonstrating OPs in enamel at a characteristic oblique angulation. In the peripheral dentine (La Fleche, Frank and addition, the content in enamel spindles was mor- Steuer, 1985; Torneck, 1994). However, the present phologically continuous with contents in dentinal conventional TEM study of samples fixed with tubules (Sharawy and Yaeger, 1986). The present aldehydes at low temperature immediately after EDS X-ray analysis of enamel spindles showed that extraction clearly demonstrated many oblique- and these structures contained less Ca and P elements cross-sectioned OPs at the DEJ, and some OPs (by wt %) than the surrounding enamel prisms, but representing either the simple projection or enamel The Extent of the Odontoblast Process 321 rod/spindle were observed invading the enamel 10) Frank RM. Etude au microscope electronique de l'donto- layer (Sigal et aL, 19841; Sigal et aL, 19842; Sigal, blaste et du canalicule dentinaire humain. Archs Oral Biol Aubin and Ten Cate, 1985;Tomeck, 1994).Further, 1966; 11: 179-199. 11) Garant PR. The orgainzation of microtubules within rat in the EDTA-demineralised specimens, a fine struc- odontoblast processes revealed by perfusion fixation with ture study revealed periodontoblastic spaces of glutaraldehyde. Archs Oral Biol 1972; 17: 1047-1058. various widths between the OP and dentinal tubu- 12) Hodson JJ. Micro-dissection and other techniques for the lar wall. These spaces contained some collagen investigation of human enamel. Brit Dent J 1952; 92: 195- fibres, amorphous substances and an electron-dense 203. 13) Holland GR. The extent of the odontoblast process in the membranous structure (lamina limitans) lining be- cat. J Anat 1976; 121: 133-149. tween the tubular wall and the OP, but no con- 14) Howard V, Reid S, Baddeley A and Boyde A. Unbiased tinuous membranous lamina limitans surrounding estimation of particle density in the tandem scanning re- the OPs was found to protrude into the enamel. flected light microscope. J Microsc 1985; 138: 203-212. Using freeze-replica TEM, we observed that most 15) Isokawa S, Yoshida M, Komura A and Iwatake Y. A preliminary study on the peritubular structure of human OPs completely filled the dentinal tubules with vir- dentinal tubules by scanning electron microscopy. J Nihon tually no space between them. We postulated that Univ Sch Dent 1972; 14: 122-125. the occurrence of large periodontoblastic spaces 16) Kelly KW, Bergenholtz G and Cox CF. The extent of the and absence of either OPs and lamina limitans in odontoblast process in Rhesus monkeys (Macaca mulatta) healthy dental tubules might be due to artefacts as observed by scanning electron microscopy. Archs Oral Biol 1981;26: 893-897. that arise during tissue processing. 17) Kodaka T, Debari K and Yamada M. Physicochemical and morphological studies of horse dentin. J Electron Microsc 1991; 40: 385-391. Acknowledgements 18) La Fleche RG, Frank RM and Steuer P. The extent of the human odontoblast process as determined by transmission electron microscopy: the hypothesis of a retractable sus- This study was performed at the Morphological pensor system. J Biol Buccale 1985; 13: 293-305. Research Facilities and Tissue Culture Research 19) Mizuhashi T and Suga S. Microradiographic investigations Facilities in the Institute of Dental Research of of carious changes in the enamel in the ground sections Osaka Dental University. We gratefully acknowl- made perpendicularly to tooth axis. Shigaku 1990; 78: 283- 312. (in Japanese) edge the assistance of laboratory technicians Mr. 20) Nakamura 0 and Kawata S. Three-dimensional transfer- Tokio Nonaka and Mr. Hideo Hori for preparing function analysis of the tomographic capability of a con- the specimens and performing the EDS analysis. focal fluorenscence microscope. J Opt Soc Am 1990; A7: 522-526. 21) Ohya K, Yasui M, Oda T, Aoki K and Ogura H. Morpho- logical observations of the hard tissues with the confocal References laser scanning microscope. Jpn J Oral Biol 1992; 34: 339- 1) AveryJK. Dentin,in: Orban'soral histology and embryol- 349. ogy,10th ed., (Bhaskar SN ed.) 101-134, Mosby, St. Louis, 22) Richard LB, Richard GK and Tung HN. Freeze fracture 1986. techniques, Plasma membrane. in: Freeze fracture images 2) BennDK and Watoson TF. Correlation between film posi- of cells and tissues. Oxford University Press, New York, tion,bite-wing shadows, clinical pitfalls, and the histologic 1991. sizeof approximal lesions. Quintessence 1989; 20: 131-141. 23) Sharawy M and Yaeger JA. Enamel. In: Orban's oral histology and embryology, 45-100, 10th ed. (Bhaskar SN 3) BoydeA and MartinL. A non-destructivesurvey of prism ed.), Mosby, St. Louis, 1986. packingpatterns in primateenamels. In: ToothEnamel IV. (Fearnhead RW and SugaS eds.)417-421, Elsevier 24) Sigal MJ, Aubin JE, Ten Cate AR and Pitaru S. The Science,Amsterdam, 1984. odontoblast process extends to the dentinoenamel junction: 4) BoydeA. Applicationof tandemscanning reflected light an immunocytochemical study of rat dentine. J Histochem microscopyand three-dimensionalimaging. Ann N.Y. Cytochem 1984'; 32: 872-877. AcedSci 1986; 483: 428-439. 25) Sigal MJ, Pitaru S, Aubin JE and Ten Cate AR. A com- 5) BradfordEW. The identity of Tomes fibre. Br DentJ 1950; bined scanning electron microscopy and immunofluore- 89:203-290. scence study demonstrating that the odontoblast process 6) BrannstrOmM and GarberoglioR. The dentinaltubules extends to the dentinoenamel junction in human teeth. andthe odontoblast processes. Acta Odont Scand 1972; 30: Anat Rec 19842;210: 453-462. 291-311. 26) Sigal MJ, Aubin JE and Ten Cate AR. An immunocyto- 7) CarlssonK, DanielssonPE, Lenz R, LiljeborgA, Map& L chemical study of the human odontoblast process using andAslund N. Three-dimensionalmicroscopy using a con- antibodies against tubulin, actin, and vimentin. J Dent Res focallaser scanning microscope. Opt Lett 1985;10: 53-55. 1985;12: 1348-1355. 8) CorpronRE and AveryJK. The ultrastructureof intra- 27) Tanaka J and Nishikawa T. Application of confocal laser dentinalnerves in developingmouse molars. Anat Rec scanning microscopy to enamel caries. Jpn J Oral Biol 1973;175: 585-606. 1995; 37: 470-480. (in Japanese) 9) FujitaT and YuasaT. KOperlicheBeobachtungen der 28) Thomas HF. The effect of various fixatives on the extent of Schmelzbiischel menschlicher Zahne. Jap J MedSc I Anat the odontoblast process in human dentine. Archs Oral Biol 1949;10: 35-42. 1983; 28: 465-469. 322 Y. Ogita et aL

29) Thomas HF and Carella P. A scanning electron microscope final tubules. Proc Roy Soc London 1856; 146: 515-522. study of dentinal tubules from un-erupted human teeth. 34) Torneck CD. Dentin-pulp complex. In: Oral histology - Archs Oral Biol 1983;28: 1125-1130. development, structure, and function -. 4th ed., (Ten Cate 30) Thomas HF and Payne RC. The ultrastructure of dentinal AR. ed.), 169-217, Mosby, St. Louis, 1994. tubules from erupted human premolar teeth. J Dent Res 35) Ueki S. Tooth removal using combination acupuncture and 1983; 62: 532-536. micro-electronic-wavestimulation - histological changes in 31) Thomas HF. The lamina limitans of human dentinal the dental pulp of deciduous teeth -. J Jpn Dent Soc Orient tubules. J Dent Res 1984;63: 1064-1066. Medic 1992; 11: 29-33. (in Japanese) 32) Thomas HF and Carella P. Correlation of scanning and 36) Weber DF and Zaki AE. Scanning and transmission elec- transmission electron microscopy of human dentinal tu- tron microscopy of tubular structures presumed to be bules. Archs Oral Biol 1984;29: 641-646. human odontoblast processes. J Dent Res 1986; 65: 982- 33) Tomes J. The presence of fibrils of soft tissue in the den- 986.

Abbreviations used in Figs

E: enamel, D: dentine, DEJ: dentino-enamel junction.

Explanation of Figures

Plate I

Fig. 1. Light microscopy of a longitudinal ground section showing dentinal tubules with their contents intruding into the enamel (arrows: enamel spindles, arrow heads: simple projections). Fig. 2. CLSM of a longitudinal ground section showing dentinal tubules containing organic substances continuous to the enamel spindle (arrow heads) with an enlarged ending (arrow) in the enamel.

Fig. 3. SEM at the dentino-enamel boundaries of an HC1-etched ground section shows odontoblast processes (arrow heads) with an enlarged ending invading the enamel, which are embedded directly in the enamel. Asterisks indicate dentinal tubules. EDS X-ray microprobe analysis was subsequently conducted on this area to obtain the results shown in Table 1.

Fig. 4. SEM photo showing an enamel rod (arrow) in the HCI-etched enamel; no hypermineralised peritubular dentine, lamina limitans or membranous sheath is found surrounding the structure. The odontoblast process intervenes between enamel (E) prisms (asterisks).

Fig. 5. Dentino-enamel boundaries of an EDTA-etched tooth. Some odontoblast processes (arrow heads) extending from dentinal tubules into the site previously occupied by the enamel are observed. An asterisk indicates a part of an enamel tuft at the dentino-enamel boundaries.

Fig. 6. Higher magnification of the dentino-enamel junction showingprojections from dentinal tubules (arrow heads) and an enamel spindle (asterisk).

Fig. 7. SEM showing divisionand union of the endings (asterisk) of the invading odontoblastic processes (arrow head); they may be fused to form a broad enamel spindle in HCI-etched ground sections. The Extent of the Odontoblast Process 323 Plate I 324 Y. Ogita et al.

Plate II

Fig. 8a—c. TEM showing odontoblast processes in the demineralised dentine. Fig. 8a. Photo of odontoblast processes (asterisks) in dentinal tubules of the middle layer dentine. Notice a bundle of collagen fibres (arrow) in the periodontoblastic space and the lamina limitans (arrow heads) lining the tubular wall. Fig. 8b. TEM of the middle layer dentine showing the electron-dense lamina limitans (arrow heads) which adheres to either the tubular wall or the cell membrane of the odontoblast process (asterisks). Some granules are found in the odontoblast process. Fig. 8c. TEM of an odontoblast process (asterisk) in the superficial dentine. Many cytoplasmic granules of various sizes and electron densities are also noted in the cell process.

Fig. 9a,b. TEM showing some odontoblast processes near the dentino-enamel boundaries. Fig. 9a. Photo showing odontoblast processes contained in superficial dentinal tubules (arrow heads). Note cell processes with a bulging ending (asterisks) at the dentino-enamel junction. Fig. 9b. Photo showing an odontoblast process (asterisk) which extends into the site originally occupied by the enamel. The Extent of the Odontoblast Process 325 Plate H 326 Y. Ogita eta!.

Plate HI

Fig. 10a,b. Freeze replica TEM of odontoblast processes in the superficial dentinal tubules. Fig. 10a. Replica showing longitudinally-sectioneddentinal tubules (arrow heads) containing freeze-fractured odontoblast pro- cesses (asterisks) in the superficial dentine and at the dentino-enamel junction. Fig. 1013. TEM showing a fractured odontoblast process with its lateral branches. The odontoblast process was freeze-fractured at the cell membrane showing both the exoplasmic (EF) and protoplasmic (PF) leaflets. The cell processes and its branch were also fractured to show their cross sections (asterisks). Note there is almost no space between the tubular wall and its associated odonto- blast process. An arrow at the lower right of the photo indicates the direction of shadowing using the vacuum evaporation technique. The Extent of the Odontoblast Process 327 Plate III