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Coronal cementogenesis in the horse
CORONAL
CEMENTOGENESIS SHEILA
Department
of Anatomy
IN THE HORSE
J. JONES and A. BOYDE
and Embryology,
University
College London,
London,
England
Summary-The enamel surface on which cementum is laid, and the development and structure of the coronal cementum of the teeth of the horse were studied by scanning electron microscopy. The enamel was completed first to the same morphological extent as that of mammalian enamel on which no cementum is laid down. However, the enamel did not become fully mature as assessed by the concentration of calcium measured by elemental X-ray analysis and furthermore partial resorption of the enamel surface preceded cementogenesis. The cementum was cellular, and was vascular where bulky, showing characteristics of both foetal and adult bone. Sharpey fibres were only incorporated in the external cementum and were usually only partly mineralized. INTRODUCTION It
has been recognized for nearly 300 yr that the enamel of equine molars is covered by cementum (Havers, 1691). The advent of improved microscopes in the last century enabled Purkinje (1835) to describe the cellularity of this tissue and its resemblance to bone. Owen (1845) also examined the histology of the coronal cementum of the horse and described the border of the enamel next to the vascular cementum in the centre of the incisor as irregularly pitted. His plate of a longitudinal section of the molar of a horse also shows an irregular junction between the enamel and the encapsulating vastmar cementum. This observation was confirmed by Kawai (1955). Although interest in coronal cementogenesis has been renewed recently (Glimcher, Freiberg and Levine, 1964; Hunt, 1959; Listgarten, 1968; Listgarten and Kamin, 1969; Longmore, 1968; Mills and Irving, 1967; Schroeder and Listgarten, 1971; Wasserman, Moskow and Rennart, 1970; Weinreb and Sharav, 1964) little attention has been paid to the enamel surface on which this cementum is laid. The purpose of this investigation was to study the enamel surface which becomes covered with cementum in the teeth of the horse, as well as the development and structure of the coronal cementum. Scanning electron microscopy was used to enable direct observation of the formative front of both the enamel and the cementum, and of the internal structure of adult cementum in fractured specimens. MATERIALS
AND
METHODS
The developing deciduous incisors and molars of four neonate horses and the functional teeth of one adult horse were fixed in IO per cent neutral form01 saline or 70 per cent ethanol. The incisors of one horse were cleansed of cellular debris by ultrasonication in a trypsin (1 per cent) and detergent solution. washed and dried from 50 per cent alcohol. Anorganic specimens were prepared from both fixed and fresh material by treatment with a 7 per cent solution of sodium hypochlorite or by extraction with hot 1.2 ethane dmmine in ;I Soxhlct apparatus. followed by thorough washing u iih \\atcr. Fiucd specimens from one ~nu~lar were
quenched in liquid Arcton 12 cooled to lust above its freeLing point ( - 155°C) by liquid nitrogen and then freeze-dried at - 70°C. All specimens were coated with carbon and gold in ULCUO and examined in a Cambridge Stereoscan scanning electron microscope (SEM) operated by 10 kV (Boyde and Wood, 1969). Stereopair micrographs with a tilt angle of 10‘ between exposures were taken to aid in the interpretation of the surfaces. Developing teeth were air dried and then embedded in epoxy resin for electron-probe X-ray emission micro-analysis. These, and unembedded specimens of functional teeth, were sectioned longitudinally and polished to obtain a flat surface. They were mounted on specimen stubs so that the top surfaces of the specimens were parallel to the surface of the stub and coated in WICUO with aluminium. Total CaKa X-ray emission with 20 kV excitation was recorded from 22 x 26.4nm fields with a constant X-ray take-off angle, using a Nuclear Diodes Inc. EDAX energy dispersive X-ray analytical system attached to the SEM. The specimen current and working distance were maintained constant throughout. The concentration of calcium in the specimens was assessed relative to a standard of compressed apatite of density 3.14, kindly supplied by Dr. J. C. Elliot. Readings of counts of the Ca peak were taken using the full width of the peak at half the maximum value (FWHM) and subtracting the background counts. At least 10 readings were made from each section; the specimens were rotated through 180 for half the readings to ensure that any minor local variation in the angle of the specimen surface would be compensated for. OBSERVATIONS
Since relatively large specimens may be mounted in the Stereoscan, several stages in the development of the enamel and coronal cementum could be observed in one tooth, and the sequence of events traced from the early stages of enamel formation to cementogenesis by moving the field of view coronally over the surface of the tooth. The present observations indicate that the enamel surface is always completed first, to an extent comparable with other mammalian teeth in which no cementurn forms on the enamel or in which cementum forms directly upon the intact enamel surface. A partial
606
Sheila J. Jones and A. Boyde
resorption of the completed enamel precedes cementum deposition in the horse. The cementum is cellular and vascular where bulky, has characteristics of both foetal and adult bone. and incorporates extrinsic Sharpey fibres on the external surfaces of the tooth.
The surface of the developing enamel changed on completion of the full thickness of the enamel. The ameloblastic (Tomes process) pits became shallower and the inter-row sheets became relatively broader and smoother. The pits sometimes showed a pattern of small bumps and depressions in their floors, and usually at the periphery which measured from 05 to I pm across (Fig. I). A granular deposit of surface enamel was produced as the final layer. Its disposition depended upon the shape of the enamel pit which is due to the final shape of the Tomes process of the ameloblast. The pits in Pattern I (Boyde. 1964, 1969) regions were shallowest, sometimes quite round, and possessed an inner circumferential ridge at the periphery of the floor of the pit (Fig. 2). Deeper pits with a steeper sloping floor also had an extra ridge in the floor but this ridge often fused with the occlusal wall of the pit and was separate from the opposite wall (Fig. 3). As the shape of the pit graded towards a Pattern 11 (Boyde, 1964, 1969), the inter-row sheets became more prominent, and the pits narrower (Fig. 4). The surface enamel appeared to be porous in anorganic specimens. It was thinnest in the troughs of the perikymata in the molars and in the incisors. In these although the surface enamel blurred the outlines of the pits, especially on the crests of the inter-row sheets (Fig. 5), the pattern could still easily be seen. The pattern on the crests of the perikymata in the molars was frequently Pattern 1 and this was in parts completely obscured by a thicker. rougher globular deposit of surface enamel (Figs. 6 and 7). The epithelial origin of the surface layer was confirmed by its presence below the ameloblasts which were retained in the freeze-dried specimens. Its texture and character were similar to that found on human permanent teeth. Large areas of defective thickness of the enamel, equivalent in area to the secretory territories of hundreds or thousands of ameloblasts. were not uncommon especially in the molars. They sometimes occurred in a group along the same developmental line (longitudinal ridge hypoplasia) or at the same developmental level (line of pits hypoplasia: Fig. 8). This hypoplasia was distinct from the pattern of the longitudinal ridges that was a feature of the enamel surface of the molars (Fig. 10). The concentration of calcium in both the developing deciduous incisors and molars of the horse compared with the apatite standard (taken as 100 per cent) was 77 per cent. This was the same as that obtained for the functional deciduous molar. Thus, the developing deciduous teeth have a relatively low level of mineralization at the time of cement deposition and this does not
increase after cementogenesis. Analysis of points along the length of the tooth from the level of initial resorption of the enamel to that covered bv a bulk of cementurn did not demonstrate any significant change in the concentration of calcium ions in the enamel in that zone. In the one functional permanent incisor which was analysed. a level of 89 per cent of the CaK8~ emission from the apatite standard was obtained. Resorption The sequence of resorption is as follows. After the completion of the enamel surface. isolated resorption lacunae were observed, usually situated along lines roughly parallel with the perikymata (Fig. 9). In the freeze-dried specimens, resorption bays were seen nestling amongst the tall ameloblasts and were sometimes filled with large cells. presumably osteoclasts. Demineralization or etching of the enamel-pit boundaries near distinct bays marked the early activity of osteoclasts (Fig. 5). Several lines of bays occurred on one surface. often hundreds of microns apart. Although the initial resorption attack was usually along a line roughly parallel with the perikymata, it did not seem to favour either the crests or the troughs. Where the resorption bays coalesced or extended to form a line. the depth and width of the resorption often appeared to show a gradient from one end of the line to the other, with isolated bays leading the process. As resorption became more widespread. its area stretched irregularly coronally and apically (Fig. 10). Wide vertical “limbs” of resorption bays occasionally crossed several perikymata. thus isolating islands of unresorbed enamel surface (Fig. I I ). The Pattern II organization of the enamel was emphasized in the resorption iacunae because the inter-row sheets and prisms etched at different rates. The inter-row sheets could be traced on to the intact surface at the side of the resorbed areas. The individual prisms of areas of etched Pattern I enamel also showed clearly. Three orders of roughness were achieved by the resorption : firstly, the gross pattern of resorption bays: secondly, the finer pattern due to the variation of etch rates of the prisms and interprismatic enamel; and thirdly, the fine porosity produced by different rates of etching of crystals or groups of crystals in the same vicinity and with the same orientation (Figs. 12-14). Except for a few deep pits, the depth of resorption was irregular and mostly shallow even where the unrepaired area was extensive. In the molars particularly, the advancing edge of the resorption unrepaired by coronal cementum was sometimes only one or two resorption lacunae wide (Fig. 15), although the incisor teeth often showed extensive areas of resorption. In one molar the enamel was entirely missing in one area and resorption of the dentine had occurred. This was again partly repaired by cementum. In the fractured specimens of a completed molar from an adult horse and in those foetal teeth where a bulk of cementurn had been deposited. the enamel surface appeared
Coronal cementogenesis in the horse to have been resorbed except for one small area in which the cementum had been fractured away, revealing the surface zone enamel apparently intact. Small areas of early cementogenesis in the foetal molars that were not surrounded by resorption bays were also noted, so that it seems that cementogenesis does sometimes occur in limited areas without prior resorption. However. some of these latter instances may also be interpreted by assuming that repair may have occurred to the limit of the resorbed areas. Areas of unresorbed enamel covered by cementum were also seen in a ground section of a developing horse molar. It was of interest that in this instance the cementum deposition was centred over a resorbed area and seemed to have “flowed” over the unresorbed enamel surface in a manner similar to that observed in human root-cementum formation where resorption into the acellular cementurn results in repair that “overflows” on to the surrounding acellular cement (Jones and Boyde. 1972).
Cementum was deposited directly on the resorbed enamel surface. The intrinsic (Jones and Boyde, 1972) collagen bundles of the cementum were arranged randomly, but parallel to the plane of the forming surface (Fig. 16). These fibre bundles ranged in diameter up to 2 Ltm, and fibrils passed from one bundle to another. Cells were abundant in the cementum from the beginning of cementogenesis, and the lacunae were about 15,rlm in their longest dimension measured parallel to the formative surface. They tended to be nearly circular at the beginning of cementogenesis when the collagen fibres were not well-orientated, and the firstformed walls of the lacunae showed a fine fibrillar collagen network (Fig. 17). The lacunae were longer and narrower towards the end of cementogenesis and had concave borders where they embraced the extrinsic fibres in the exterior cementum of the molars. Canaliculi were abundant and measured from 0.1 to 0.3 pm in diameter. In the thicker cementum, the lacunae near vascular channels were sometimes arranged with their long axes on an arc around the vessel. similar to those of primary osteones. In anorganic specimens, the first signs of repair were due to the mineral phase deposited within the “intrinsic” cement-matrix collagen fibres. These appeared as discrete calcified aggregates. either small and spherical or larger and spindle-shaped and presumably aligned along the axes of the collagen fibres. Specimens treated briefly with trypsin showed a partial solubility of the collagen fibrils similar to that exhibited by foetal bone (Boyde. 1972; Boyde and Jones, 1972). In these specimens and in those treated with sodium hypochlorite for a short time, the mineralization during early cementogenesis showed as nodules within the fibre bundles (Fig. 18). In areas where the mineralization was more complete, and the organic matrix completely removed, the mineralized segments were spindleshaped and about I ,nm long (Fig. 19). The back walls of the half-formed ccmentocyte lacunae now showed a
607
fully mineralized fine fibrillar meshwork (Fig. 20). At the completion of cementogenesis in the exterior cementum, a fully mineralized front resulted (Fig. 21). The calcification of the intrinsic component of the cementum generally remained incomplete in the bulk of the cementum. The deposition of the cementum followed the distribution of resorbed areas and the tissue rapidly acquired a considerable thickness. Deep pits or clefts remained in the cementum in those areas where resorption of the enamel had not occurred (Fig. 22). Tracing the cementogenesis occlusally, these deficiencies grew fewer and were finally obliterated in those specimens with a considerable bulk of cementum over the tips of the cusps. Vascular loops incorporated in the cementum had a diameter of 25-50 pm and radiated out from the enamel to the cementum surface with their centres ranging from 100 to 200 pm apart (Fig. 23). In the clinical crown of functioning cheek teeth. the vascular channels were either empty or filled with a mineralized substance. Sharpey-fibre bundles could always be recognized in the forming mineral surface, even where the level and pattern of mineralization and the fibre size in the extrinsic and intrinsic fibres were the same. because their orientation was distinctive. Sharpey fibres with a diameter of 5- IO pm were included in the external coronal cementum after a layer of cementum with intrinsic fibres only had been laid down. The sites of insertion of the as yet unmineralized extrinsic fibre bundles were represented as holes in anorganic specimens. Mineralization of the Sharpeyfibre bundles could be seen in the depths of the holes. In later stages of mineralization, the centres often remained uncalcified even in otherwise well-mineralized surfaces (Fig. 21) until finally mineralization proceeded along the extrinsic fibres within the surface layer up to and then beyond the completed intrinsic cementum surface. In fractured anorganic specimens, the pattern of the spread of mineralization along the Sharpey fibres was plain. Discrete centres of mineral deposition occurred along the lengths of the collagen fibres that constituted the extrinsic fibre bundle. These fibres were about 2 pm in diameter and the centres of mineralization some 335 pm apart. A finer striation due to the mineralization of the individual fibrils was obvious on the surface of the mineralized segments of the fibres. The centres of calcification extended and became spindle-shaped with the long axis of the spindle along the length of the Iibre. Most spindles fused with those on either side in the same fibre; some, however, remained separate even within the depth of the cementum. Mineralization of the fibre was generally incomplete; thus the mineralized spindles. although fused, were constricted at the sites of fusion, giving a beaded appearance (Fig. 24). Even where the fibres were fully mineralized at the periphery of the bundle, those more centrally located were either unmineralized or incompletely mineralized. After pursuing an through the cementum. the undulating course
Sheila J. Jones and A. Boyde
608
Sharpey-fibres left the surface either at an angle with the fibres pointing occlusally, or perpendicular to the surface. The level of mineralization of the extrinsic fibre bundle was perpendicular to the long axis of the bundle, irrespective of its angle of insertion, and the plane of the mineral front of the intrinsic matrix. DM‘I’SSION
Before the development of the scanning electron microscope, it was not possible to view surfaces directly in such a wide range of magnifications. Furthermore. to build ;I composite picture from sections is a tedious process, especially where enamel is concerned. This may explain why the phenomenon of the resorption of the enamel surface prior to the deposition of coronal cementum has not previously been described in any great detail in the horse or in any other species. With the benefit of hindsight, the pattern of cementum deposition and resorption described with the SEM can be seen quite clearly on anorganic specimens using a stereobinocular light microscope. especially after coating the surface with carbon and gold. Resorption of the enamel of equine teeth was recorded by Owen (1845).He studied longitudinal sections of the teeth and measured the hemispherical pits he observed beneath the cementum. Owen’s tinding was confirmed by Kawai (1955) who noted resorption bays in ground sections of an equine molar. and observed that the connection between the enamel and the coronal cementum was thereby improved as the surface was enlarged. Resorption of the enamel of the deciduous incisors of the Indian elephant prior to coronal cementogenesis was recorded by Riise (I 894). He examined forming molar teeth also. but did not describe a similar resorption in them. Kauai (1955), however. believed that the molar enamel of the Indian elephant was subject to resorption. It is difficult to discern any evidence for this observation in his figure of the elephant molar section ( x 50 magnification) although the enamel-cementum interface is rough. Schmidt and Keil (1971) found that the enamel under the cementum in one deciduous molar of an African elephant had been partly resorbed. We found no resorption of the enamel before cmnenturn deposition in a parallel investigation of coronal cementogenesis in the molars of the rabbit, some rodents and bovid species and the elephant (Jones and Boyde, unpublished observations). It might possibly be considered that the condition in the horse was pathological: however, it was observed in the developing deciduous molars and upper incisor pits of all four neonates in this study, and in mature functional teeth. Resorption of enamel is known to be an occasional feature of prolonged non-eruption of human teeth (Kronfeld. 1938a: Franklin, 1972). More commonly. such teeth are covered by cementum without prior resorption of the cn:unel (Kronfeld. 19333). Early cessation of the acti\it! of amcloblastc in hypoplastic
areas of human teeth (Boyde. 1970) does not attract osteoclastic activity either. In one horse molar examined in the present study. the enamel was totally missing from an area of the crown and resorption of the dentine had cxxurrcd. Thk Inay have been due to exceptionally carI> dcgcncr~~t~on or lack of differentiation of the enamel epithelium in that area. Thus large deep pits in the enamel based b! cementum may have been enamel-fret originally. The problem of what prompts this physiological osteoclastic activity in the horse is puzzling. The larger areas of unrepaired resorption bays in the horse incisor compared with the molars may he related to a faster rate of formation of the incisors at ;I similar dcvelopmental stage. In the region of the ucll-marked perikymata of the molars. the sequence of developmental stages would be compressed into a more limited length of the tooth. The osteoclastic activity is brief: the pattern of bands and rings of resorption bays suggests the existence of waves of rcsorptibc actil‘ity moving 0ve1 the surface. lt is impossible to sa> from this stud) whether the rcsorptive cells themselves move on across the surface to fresh fields. or whether. after a brief activity. they die or change their function whilst a new lint of cells takes up the process of resorption. Osteociasts resorbing bones are very active mobile cells. Although they remain vital for only 2 or 3 dabs m tissue culture (Hancox. 1972). this may not ret&t their life irk I>~IYJ. The significance of the process of resorption of the enamel before cement deposition is casicr to guess. The surface area for the attachment of the coronal cetncnturn is increased in three ways: firstly because of the resorption bays, secondly by the roughness due to the different rate of etching of the prism hodie> and the inter-rowsheetinterprismatic arcas. and thirdly hecausc of the variation in the rate of etching of single crystals, or groups of crystals. which products a tint porosity ol the surface. Since the adhesion of the cementurn to enamel would be enhanced at all three levels of roughness bq the interlocking of the tissues. one wonders why such resorption is not encountered in other species. Of coronal cementogcncsis in the othet mammals which wc have invcstigatsd. that of the African elephant is the most like. A large bulk 01 vascular and cellular ccmentum is laid down rapidly on the enamel of the molars in a similar manner to the equine teeth but, although irregularities and dcficienties in the enamel surface were commonly found in the molars, resorption bays were not. The fine pattern of resorption been in the horse enamel is very similar to the etch pattern produced b> acids under laboratory conditions.Tuo levels of rctcntion only are obtained by such etching: firstly that due to the three-dimensional pattern produced by the var\ing orientation of the crystals in the prlsims and hence their different rates of solution. and secondly the finer pattern produced bt uneven etching rates of the crystals with similar orientation which products a porosity of significant depth (Ho~dc. Jonch and Reynolds. unpublished).
Coronal cementogenesis in the horse Resorption of the enamel surface in the horse does not follow immediately upon completion of the full thickness of the tissue. ft is probable, therefore, that the enamel has time partly to mature before resorption and cementum formation occur. Listgarten (1968) found that none of the enamel in the developing bovine molars he examined was fully mineralized before cementum was deposited on the surface. It is unlikely that the enamel could continue to mature beneath the cementurn because such maturation would involve not only the deposition of more mineral, but also the withdrawal of water and organic matrix. The X-ray emission micro-analysis data from the horse teeth conhrmed that no further maturation occurs after cementogenesis in the deciduous teeth, and suggested that the level of mineralization attained in the permanent incisor was initially considerably higher than that in the other teeth. It is debatable whether the degree of mineralization reached by the enamel affects either the rate or the extent of the subsequent resorption. The granular surface layer of equinine enamel could be a combination of the final secretion of the ameloblasts and organic matrix withdrawn from the immature enamel and then mineralized without being absorbed by the ameloblasts. This might explain the nodular. rough layer on the crests of the perikymata of some molars. Functionally. this might be significant in increasing the mechanical interlocking between enamel and cementum in any areas which are not resorbed prior to cementum deposition. The pattern of mineralization of the “intrinsic” matrix fibres of the cementum varies from one similar to that of primary bone (Fig. 18 : see also Boyde, 1972) to that of adult lamcllar bone (Figs. 19 and 20). We speculate that this is due to ditferences in the rate of formation and mineralization of the cementurn. +!,PUN /~,r/Hr,irc,,ll.s~~This work has been supported by the Medical Research Council and the Science Research Council. WC arc grateful to P. Reynolds and Elaine Bailey for technical assistance.
REFERENCES
Boydc A. 1964. The structure and development of mammalian enamel. Ph.D. Thesis, University of London. Boyde A. 1969. Correlation of ameloblast size with enamel prism pattern: use of scanning electron microscope to make surface area measurements. Z. Zellforsch. 93, 583593. Boy& A. 1970. The surface of enamel in human hypoplastic teeth. .4rchs orul Biol. 1.5, 897-898. Boyde A. 1972. Scanning electron microscope studies of hone In: Tin Biocherni.str.r und Physiology ofBone (edited by Bournc G. H.), 2nd Edn, Vol. I. Academic Press-New York.
609
Boyde A. and Jones S. J. 1972. Scanning electron microscope studies of the formation of mineralized tissues. In: Droe/oprttmtu~ Aspects ofOral Biology (edited by Slavkin H. C. and Bavetta L. A.). Academic Press, New York, Boyde A. and Wood C. 1969. Preparation of animal tissues for surface-scanning electron microscopy. J. Microsc. 90, 22 l--249. Franklin C. D. 1972. Ankylosis of an unerupted third molar by inostosis of enamel. Br. dmr. J. 133, 346-347. Glimcher M. J., Freiberg U. A. and Levine P. T. 1964. The identification and characterization of a calcified layer of coronal cementum in erupted bovine teeth. J. u/tr.trsttwt. Rcs. IO, 76-88. Hancox N. M. 1972. The ostcoclasts. In: 7‘hcBio~,/l[,/tri.r/,,l, md I’h\..\io/o~q,v of Bow (edited b! Bourns G. H.). 2nd Edn. Vol. 1. (‘hap. 3. Academic Press. New York. Havcrs C‘. 1691. 5th discourse on hones and tocth IO (he Royal Society (1689) In: Ostroiogiu Nocu 01 Soare ,%‘en Ohsrwutions of the Bones. Smith, London. Hunt A. M. 1959. A description of the molar teeth and investing tissues of normal guinea pigs. J. dent. Rex 38, 216231. Jones S. J. and Boyde A. 1972. A study of human root cementum surfaces as prepared for and examined in the scanning electron microscope. Z. Zr&r.xh. 130, 318338. Kawai N. 1955. Comparative anatomy of the bands of Schreger. Okujimas Fo/. mat. jap. 27, I 15-~13 1. Kronfeld R. 1938a. Coronal cementum and coronal resorption. J. dent. Rex 17, 151-159. Kronfeld R. 1938b. The biology of cementum. .I. 4~1. drrrf. Ass. 25, 1451~1461. Listgarten M. A. 1968. A light and electron microscopic study of coronal cementogenesis. Archs ortrl Biol. 13. 93114. Listgarten M. A. and Kamin A. 1969. The development of a cementurn layer over the enamel surface of rabbit molars-a light and electron microscopic study. Arch\ orul Biol. 14, 961-985. Longmore B. 1968. Coronal cementum in the rabbit. guinea pig and the horse. J. dent. Rrs. 47,997 -998. Mills P. B. and Irving J. T. 1967. Coronal cementogenesis in cattle, 4rch\ orrrl Rio/. 12, 929- 932. Neubauer C. and Schnitger A. 1970. Fin ncues Betrachtungsgerat fur Stereohildpaare aller Formrte. Bcitr. elektronen mikrosop. Direkruhh. Ohcr$. 3,41 I 41-I. Owen R. 1845. Odontograph~~. Balhere. London. Purkinje J. E. 1835. In: Fraenkel M. De Prnitori Drntiurn Hummorw~ Strucfurcr Oh.wrrc~tionrv. Inaug. Drss. Breslau. Rose C. 1894. iiber den Zahnbau und den Zahnwechsel von Elephas indicus. Molphol.
Schroeder
ofthe
4rh. 3, 173 194.
H. E. and Listgarten Dewloping
Epitheliul
M. A. 1971. Attuchrnent
FineSt,uctuw
of’ Humw~
7&h.
Karger, Base]. Schmidt W. J. and Keil A. 1971. Polurkin~g mi~w~.scvp!’ of Denrul Ti.ssw,s. Pergamon Press, Oxford. Wasserman B. H., Moskow B. S. and Rennert M. C. 1970. Dental anatomy and corona1 cementum m the Mongolian gerbil. J. pwiodont. Rex 5, 208-218. Weinreb M. M. and Sharav Y. 1964. Tooth development in sheep. 4rn. J. err Rm. 25, 89 l-908.
610
Sheila J. Jones and A. Boyde R&umC --La surface de I’~ma11. recouverte dc cement. ainsi que lc dt;\eloppemcnt ct la structure du cCment coronaire des dents de cheval sont btudits par microscopic Clectronique par balay,age. L.&mail c\t tout d’abord form6 de la mPme faqon que dans I’Cmail de mammif&e, chcL lcqucl 11 n’y a pas dc formation de cement. Cependant I’Cmail ne subit pas unc maturation cornpI&. SI I’on en Jugc par la concentration en calcium mesurCe par l‘analyse CltYmcntaire cn rayons X. Dc plus. une r?sorption partielle de la surface de I’t:mail prCctde la c6mcntogen&e. Le ci:mcnt c
Figs.
I-20
inclusive
and
Fig. 22 show neonate horse deciduous teeth: erupted teeth from a young adult horse.
Figures
21. 23 and
24 show
Fig. I. Tomes process pits at completion of the full enamel thickness with bumps and deprcsaions their floors and. to a lesser extent, on the ridges between the pits. Field width 20 Itm. Fig. 2. Pattern
I enamel pits in deciduous
molar with a circumferential of the pit. Field width I I llrn.
Fig. 3. Stereopair. Fig. 4. Pattern
Deciduous
II alignment
molar
ridge at the periphery
in
of the floor
pits with sloping floors m which the extra ridge has fused with the occlusal wall. Field width I I /rm_
of narrow.
deep pits. Field uidth
20 ,trn
Fig. 5. Surface enamel has blurred the crests of the inter-row sheets. Parts of three trcsorption left and early etching of the surface at lower ccntrc. Field width 50 ~cm.
bays arc at
Coronal cementogenesis
in the horse
PLATE I
A.O.B.
f.p. 610
SHEILA J.JONES and A. BOYDE
PLATE 2
Coronal
cementogenesis
in the horse
611
PLATE 2 Fig. 6. Rough globular
deposition on crest of perikymata above contrasts with the thin deposit enamel in the trough. Deciduous molar. Field width 100 pm.
Fig. 7. Pattern II enamel in the troughs and Pattern I enamel partly obscured deposit of surface enamel on the crests of the perikymata of a deciduous molar. Fig. 8. Large hypoplastic Fig. 9. Circular
resorption
defects in the enamel at approximately Field width 500 itm. bay in enamel
surface.
Parts
Fig. 10. Resorbing areas on molar enamel. Small isolated is ridged longitudinally, as well as horizontally Fig.
11. Island of unresorbed
enamel
by granular, globular Field width 240 pm.
the same developmental
of two others
of surface
level in a molar
at top right. Field width 50 pm.
pits can be seen in lower part. The molar surface by the perikymata. Field width 1 mm.
ringed by resorption bays in area specimen. Field width 745 pm.
of cementogenesis.
Anorganic
Sheila J. Jones and A. Boyde
Fig. 12. Junction 01‘ resorbed cnCimcl (ahove) and cemcntogenesls (hrloti 1. The pattern of the cnamcl ia shov,n 111the 1x1~ due tu the dill’crcncc m the etch rates 01 the prism boundary and ccntrc. The stcrcopair illustrates
the depth
of the
prismatic
resorption
hays.
viewer (Neuhauer
(This
pair
and Schnitger.
should
he viewed
with
1970).) Field width
a System
46 I’m.
Nesh
type
Coronal cementogenesis
in the horse
PLA
.TE 3
A .O.B. f.p. 612
SHEILA J. JONES
PLATE 4
and
A. BOYD~
Coronal
cementogenesis
in the horse
613
PI A,,. 4 Fig. 13. Rough,
irregular
Fig. 14. Three orders
surface produced by resorption of enamel. Prism different rates of resorption. Field width 100 pm.
of roughness:
due to the bays. the prisms and the crystals.
structure
emphasized
by
Field width 20 pm.
Fig. IS. Narrow advancing edge of resorption with bays with their long axes at right angles to the direction of advance. Cementogenesis is repairing the resorption in right half of the picture. Unresorbed enamel at left. Anorganic specimen. Field width 240 pm. Fig. 16. Randomly Fig. 11.Random Fig. 18. Pattern
arranged
collagen
collagen
fibres in the plane of the forming 59 /Irn.
fibrils in back wall of half-formed
cementum
lacuna in cementurn
surface.
Field width
surface. Field width 24 ,um.
of mineralization of cementum with small mineral particle aggregates. at centre. Anorganic specimen. Field width 55 pm.
Cementocyte
lacuna
hl4
Sheila J. Jones and A. Boyde
t tg. 19. Later stage of mineralization Three cementocyte lacunae Fig. 20. Centre
lacuna
with larger spindle-shaped mineralized at centre horizon. Anorganic specimen,
of Fig. 19. showing
fine fibriikdr meshwork width 24 pm.
segments of collagen Field width 72 pm.
of fully mineralized
tibres.
back waif. Fiefd
Fig. 21. Completion of cementogencsis: the openings of the canaliculi are obvious in the fully mineralized surface. Cementocyte lacuna at centre is bordered by two Sharpey fibres which are not completely mineralized. Anorganic specimen. Field width 25 pm. k’ig. 22. Clefts or pits remain
in the cementum where resorption of the enamel Occlusal to bottom right. Field width 5 mm.
has not yet occurred.
Fig. 23. Surface of erupted molar: scratches due to abrasion cover the cementum. Vascular openings are regularly placed at the bases of hollows. Many cementocyte lacunae are exposed at the surface. Field width 420 pm. Fig. 24. Fractured anorgamc ccmentum. The intrinsic tibres are not well mineralized, and the Sharpey tibres running from top left to bottom right have unmineralized cores and a constricted pattern of fibre mineralization. Field width 50 pm.
Coronal
cementogenesis
in the horse
PLATE 5 A.O.R. f.p. 614
CEMENTOGENESIS SHEILA
Department
of Anatomy
IN THE HORSE
J. JONES and A. BOYDE
and Embryology,
University
College London,
London,
England
Summary-The enamel surface on which cementum is laid, and the development and structure of the coronal cementum of the teeth of the horse were studied by scanning electron microscopy. The enamel was completed first to the same morphological extent as that of mammalian enamel on which no cementum is laid down. However, the enamel did not become fully mature as assessed by the concentration of calcium measured by elemental X-ray analysis and furthermore partial resorption of the enamel surface preceded cementogenesis. The cementum was cellular, and was vascular where bulky, showing characteristics of both foetal and adult bone. Sharpey fibres were only incorporated in the external cementum and were usually only partly mineralized. INTRODUCTION It
has been recognized for nearly 300 yr that the enamel of equine molars is covered by cementum (Havers, 1691). The advent of improved microscopes in the last century enabled Purkinje (1835) to describe the cellularity of this tissue and its resemblance to bone. Owen (1845) also examined the histology of the coronal cementum of the horse and described the border of the enamel next to the vascular cementum in the centre of the incisor as irregularly pitted. His plate of a longitudinal section of the molar of a horse also shows an irregular junction between the enamel and the encapsulating vastmar cementum. This observation was confirmed by Kawai (1955). Although interest in coronal cementogenesis has been renewed recently (Glimcher, Freiberg and Levine, 1964; Hunt, 1959; Listgarten, 1968; Listgarten and Kamin, 1969; Longmore, 1968; Mills and Irving, 1967; Schroeder and Listgarten, 1971; Wasserman, Moskow and Rennart, 1970; Weinreb and Sharav, 1964) little attention has been paid to the enamel surface on which this cementum is laid. The purpose of this investigation was to study the enamel surface which becomes covered with cementum in the teeth of the horse, as well as the development and structure of the coronal cementum. Scanning electron microscopy was used to enable direct observation of the formative front of both the enamel and the cementum, and of the internal structure of adult cementum in fractured specimens. MATERIALS
AND
METHODS
The developing deciduous incisors and molars of four neonate horses and the functional teeth of one adult horse were fixed in IO per cent neutral form01 saline or 70 per cent ethanol. The incisors of one horse were cleansed of cellular debris by ultrasonication in a trypsin (1 per cent) and detergent solution. washed and dried from 50 per cent alcohol. Anorganic specimens were prepared from both fixed and fresh material by treatment with a 7 per cent solution of sodium hypochlorite or by extraction with hot 1.2 ethane dmmine in ;I Soxhlct apparatus. followed by thorough washing u iih \\atcr. Fiucd specimens from one ~nu~lar were
quenched in liquid Arcton 12 cooled to lust above its freeLing point ( - 155°C) by liquid nitrogen and then freeze-dried at - 70°C. All specimens were coated with carbon and gold in ULCUO and examined in a Cambridge Stereoscan scanning electron microscope (SEM) operated by 10 kV (Boyde and Wood, 1969). Stereopair micrographs with a tilt angle of 10‘ between exposures were taken to aid in the interpretation of the surfaces. Developing teeth were air dried and then embedded in epoxy resin for electron-probe X-ray emission micro-analysis. These, and unembedded specimens of functional teeth, were sectioned longitudinally and polished to obtain a flat surface. They were mounted on specimen stubs so that the top surfaces of the specimens were parallel to the surface of the stub and coated in WICUO with aluminium. Total CaKa X-ray emission with 20 kV excitation was recorded from 22 x 26.4nm fields with a constant X-ray take-off angle, using a Nuclear Diodes Inc. EDAX energy dispersive X-ray analytical system attached to the SEM. The specimen current and working distance were maintained constant throughout. The concentration of calcium in the specimens was assessed relative to a standard of compressed apatite of density 3.14, kindly supplied by Dr. J. C. Elliot. Readings of counts of the Ca peak were taken using the full width of the peak at half the maximum value (FWHM) and subtracting the background counts. At least 10 readings were made from each section; the specimens were rotated through 180 for half the readings to ensure that any minor local variation in the angle of the specimen surface would be compensated for. OBSERVATIONS
Since relatively large specimens may be mounted in the Stereoscan, several stages in the development of the enamel and coronal cementum could be observed in one tooth, and the sequence of events traced from the early stages of enamel formation to cementogenesis by moving the field of view coronally over the surface of the tooth. The present observations indicate that the enamel surface is always completed first, to an extent comparable with other mammalian teeth in which no cementurn forms on the enamel or in which cementum forms directly upon the intact enamel surface. A partial
606
Sheila J. Jones and A. Boyde
resorption of the completed enamel precedes cementum deposition in the horse. The cementum is cellular and vascular where bulky, has characteristics of both foetal and adult bone. and incorporates extrinsic Sharpey fibres on the external surfaces of the tooth.
The surface of the developing enamel changed on completion of the full thickness of the enamel. The ameloblastic (Tomes process) pits became shallower and the inter-row sheets became relatively broader and smoother. The pits sometimes showed a pattern of small bumps and depressions in their floors, and usually at the periphery which measured from 05 to I pm across (Fig. I). A granular deposit of surface enamel was produced as the final layer. Its disposition depended upon the shape of the enamel pit which is due to the final shape of the Tomes process of the ameloblast. The pits in Pattern I (Boyde. 1964, 1969) regions were shallowest, sometimes quite round, and possessed an inner circumferential ridge at the periphery of the floor of the pit (Fig. 2). Deeper pits with a steeper sloping floor also had an extra ridge in the floor but this ridge often fused with the occlusal wall of the pit and was separate from the opposite wall (Fig. 3). As the shape of the pit graded towards a Pattern 11 (Boyde, 1964, 1969), the inter-row sheets became more prominent, and the pits narrower (Fig. 4). The surface enamel appeared to be porous in anorganic specimens. It was thinnest in the troughs of the perikymata in the molars and in the incisors. In these although the surface enamel blurred the outlines of the pits, especially on the crests of the inter-row sheets (Fig. 5), the pattern could still easily be seen. The pattern on the crests of the perikymata in the molars was frequently Pattern 1 and this was in parts completely obscured by a thicker. rougher globular deposit of surface enamel (Figs. 6 and 7). The epithelial origin of the surface layer was confirmed by its presence below the ameloblasts which were retained in the freeze-dried specimens. Its texture and character were similar to that found on human permanent teeth. Large areas of defective thickness of the enamel, equivalent in area to the secretory territories of hundreds or thousands of ameloblasts. were not uncommon especially in the molars. They sometimes occurred in a group along the same developmental line (longitudinal ridge hypoplasia) or at the same developmental level (line of pits hypoplasia: Fig. 8). This hypoplasia was distinct from the pattern of the longitudinal ridges that was a feature of the enamel surface of the molars (Fig. 10). The concentration of calcium in both the developing deciduous incisors and molars of the horse compared with the apatite standard (taken as 100 per cent) was 77 per cent. This was the same as that obtained for the functional deciduous molar. Thus, the developing deciduous teeth have a relatively low level of mineralization at the time of cement deposition and this does not
increase after cementogenesis. Analysis of points along the length of the tooth from the level of initial resorption of the enamel to that covered bv a bulk of cementurn did not demonstrate any significant change in the concentration of calcium ions in the enamel in that zone. In the one functional permanent incisor which was analysed. a level of 89 per cent of the CaK8~ emission from the apatite standard was obtained. Resorption The sequence of resorption is as follows. After the completion of the enamel surface. isolated resorption lacunae were observed, usually situated along lines roughly parallel with the perikymata (Fig. 9). In the freeze-dried specimens, resorption bays were seen nestling amongst the tall ameloblasts and were sometimes filled with large cells. presumably osteoclasts. Demineralization or etching of the enamel-pit boundaries near distinct bays marked the early activity of osteoclasts (Fig. 5). Several lines of bays occurred on one surface. often hundreds of microns apart. Although the initial resorption attack was usually along a line roughly parallel with the perikymata, it did not seem to favour either the crests or the troughs. Where the resorption bays coalesced or extended to form a line. the depth and width of the resorption often appeared to show a gradient from one end of the line to the other, with isolated bays leading the process. As resorption became more widespread. its area stretched irregularly coronally and apically (Fig. 10). Wide vertical “limbs” of resorption bays occasionally crossed several perikymata. thus isolating islands of unresorbed enamel surface (Fig. I I ). The Pattern II organization of the enamel was emphasized in the resorption iacunae because the inter-row sheets and prisms etched at different rates. The inter-row sheets could be traced on to the intact surface at the side of the resorbed areas. The individual prisms of areas of etched Pattern I enamel also showed clearly. Three orders of roughness were achieved by the resorption : firstly, the gross pattern of resorption bays: secondly, the finer pattern due to the variation of etch rates of the prisms and interprismatic enamel; and thirdly, the fine porosity produced by different rates of etching of crystals or groups of crystals in the same vicinity and with the same orientation (Figs. 12-14). Except for a few deep pits, the depth of resorption was irregular and mostly shallow even where the unrepaired area was extensive. In the molars particularly, the advancing edge of the resorption unrepaired by coronal cementum was sometimes only one or two resorption lacunae wide (Fig. 15), although the incisor teeth often showed extensive areas of resorption. In one molar the enamel was entirely missing in one area and resorption of the dentine had occurred. This was again partly repaired by cementum. In the fractured specimens of a completed molar from an adult horse and in those foetal teeth where a bulk of cementurn had been deposited. the enamel surface appeared
Coronal cementogenesis in the horse to have been resorbed except for one small area in which the cementum had been fractured away, revealing the surface zone enamel apparently intact. Small areas of early cementogenesis in the foetal molars that were not surrounded by resorption bays were also noted, so that it seems that cementogenesis does sometimes occur in limited areas without prior resorption. However. some of these latter instances may also be interpreted by assuming that repair may have occurred to the limit of the resorbed areas. Areas of unresorbed enamel covered by cementum were also seen in a ground section of a developing horse molar. It was of interest that in this instance the cementum deposition was centred over a resorbed area and seemed to have “flowed” over the unresorbed enamel surface in a manner similar to that observed in human root-cementum formation where resorption into the acellular cementurn results in repair that “overflows” on to the surrounding acellular cement (Jones and Boyde. 1972).
Cementum was deposited directly on the resorbed enamel surface. The intrinsic (Jones and Boyde, 1972) collagen bundles of the cementum were arranged randomly, but parallel to the plane of the forming surface (Fig. 16). These fibre bundles ranged in diameter up to 2 Ltm, and fibrils passed from one bundle to another. Cells were abundant in the cementum from the beginning of cementogenesis, and the lacunae were about 15,rlm in their longest dimension measured parallel to the formative surface. They tended to be nearly circular at the beginning of cementogenesis when the collagen fibres were not well-orientated, and the firstformed walls of the lacunae showed a fine fibrillar collagen network (Fig. 17). The lacunae were longer and narrower towards the end of cementogenesis and had concave borders where they embraced the extrinsic fibres in the exterior cementum of the molars. Canaliculi were abundant and measured from 0.1 to 0.3 pm in diameter. In the thicker cementum, the lacunae near vascular channels were sometimes arranged with their long axes on an arc around the vessel. similar to those of primary osteones. In anorganic specimens, the first signs of repair were due to the mineral phase deposited within the “intrinsic” cement-matrix collagen fibres. These appeared as discrete calcified aggregates. either small and spherical or larger and spindle-shaped and presumably aligned along the axes of the collagen fibres. Specimens treated briefly with trypsin showed a partial solubility of the collagen fibrils similar to that exhibited by foetal bone (Boyde. 1972; Boyde and Jones, 1972). In these specimens and in those treated with sodium hypochlorite for a short time, the mineralization during early cementogenesis showed as nodules within the fibre bundles (Fig. 18). In areas where the mineralization was more complete, and the organic matrix completely removed, the mineralized segments were spindleshaped and about I ,nm long (Fig. 19). The back walls of the half-formed ccmentocyte lacunae now showed a
607
fully mineralized fine fibrillar meshwork (Fig. 20). At the completion of cementogenesis in the exterior cementum, a fully mineralized front resulted (Fig. 21). The calcification of the intrinsic component of the cementum generally remained incomplete in the bulk of the cementum. The deposition of the cementum followed the distribution of resorbed areas and the tissue rapidly acquired a considerable thickness. Deep pits or clefts remained in the cementum in those areas where resorption of the enamel had not occurred (Fig. 22). Tracing the cementogenesis occlusally, these deficiencies grew fewer and were finally obliterated in those specimens with a considerable bulk of cementum over the tips of the cusps. Vascular loops incorporated in the cementum had a diameter of 25-50 pm and radiated out from the enamel to the cementum surface with their centres ranging from 100 to 200 pm apart (Fig. 23). In the clinical crown of functioning cheek teeth. the vascular channels were either empty or filled with a mineralized substance. Sharpey-fibre bundles could always be recognized in the forming mineral surface, even where the level and pattern of mineralization and the fibre size in the extrinsic and intrinsic fibres were the same. because their orientation was distinctive. Sharpey fibres with a diameter of 5- IO pm were included in the external coronal cementum after a layer of cementum with intrinsic fibres only had been laid down. The sites of insertion of the as yet unmineralized extrinsic fibre bundles were represented as holes in anorganic specimens. Mineralization of the Sharpeyfibre bundles could be seen in the depths of the holes. In later stages of mineralization, the centres often remained uncalcified even in otherwise well-mineralized surfaces (Fig. 21) until finally mineralization proceeded along the extrinsic fibres within the surface layer up to and then beyond the completed intrinsic cementum surface. In fractured anorganic specimens, the pattern of the spread of mineralization along the Sharpey fibres was plain. Discrete centres of mineral deposition occurred along the lengths of the collagen fibres that constituted the extrinsic fibre bundle. These fibres were about 2 pm in diameter and the centres of mineralization some 335 pm apart. A finer striation due to the mineralization of the individual fibrils was obvious on the surface of the mineralized segments of the fibres. The centres of calcification extended and became spindle-shaped with the long axis of the spindle along the length of the Iibre. Most spindles fused with those on either side in the same fibre; some, however, remained separate even within the depth of the cementum. Mineralization of the fibre was generally incomplete; thus the mineralized spindles. although fused, were constricted at the sites of fusion, giving a beaded appearance (Fig. 24). Even where the fibres were fully mineralized at the periphery of the bundle, those more centrally located were either unmineralized or incompletely mineralized. After pursuing an through the cementum. the undulating course
Sheila J. Jones and A. Boyde
608
Sharpey-fibres left the surface either at an angle with the fibres pointing occlusally, or perpendicular to the surface. The level of mineralization of the extrinsic fibre bundle was perpendicular to the long axis of the bundle, irrespective of its angle of insertion, and the plane of the mineral front of the intrinsic matrix. DM‘I’SSION
Before the development of the scanning electron microscope, it was not possible to view surfaces directly in such a wide range of magnifications. Furthermore. to build ;I composite picture from sections is a tedious process, especially where enamel is concerned. This may explain why the phenomenon of the resorption of the enamel surface prior to the deposition of coronal cementum has not previously been described in any great detail in the horse or in any other species. With the benefit of hindsight, the pattern of cementum deposition and resorption described with the SEM can be seen quite clearly on anorganic specimens using a stereobinocular light microscope. especially after coating the surface with carbon and gold. Resorption of the enamel of equine teeth was recorded by Owen (1845).He studied longitudinal sections of the teeth and measured the hemispherical pits he observed beneath the cementum. Owen’s tinding was confirmed by Kawai (1955) who noted resorption bays in ground sections of an equine molar. and observed that the connection between the enamel and the coronal cementum was thereby improved as the surface was enlarged. Resorption of the enamel of the deciduous incisors of the Indian elephant prior to coronal cementogenesis was recorded by Riise (I 894). He examined forming molar teeth also. but did not describe a similar resorption in them. Kauai (1955), however. believed that the molar enamel of the Indian elephant was subject to resorption. It is difficult to discern any evidence for this observation in his figure of the elephant molar section ( x 50 magnification) although the enamel-cementum interface is rough. Schmidt and Keil (1971) found that the enamel under the cementum in one deciduous molar of an African elephant had been partly resorbed. We found no resorption of the enamel before cmnenturn deposition in a parallel investigation of coronal cementogenesis in the molars of the rabbit, some rodents and bovid species and the elephant (Jones and Boyde, unpublished observations). It might possibly be considered that the condition in the horse was pathological: however, it was observed in the developing deciduous molars and upper incisor pits of all four neonates in this study, and in mature functional teeth. Resorption of enamel is known to be an occasional feature of prolonged non-eruption of human teeth (Kronfeld. 1938a: Franklin, 1972). More commonly. such teeth are covered by cementum without prior resorption of the cn:unel (Kronfeld. 19333). Early cessation of the acti\it! of amcloblastc in hypoplastic
areas of human teeth (Boyde. 1970) does not attract osteoclastic activity either. In one horse molar examined in the present study. the enamel was totally missing from an area of the crown and resorption of the dentine had cxxurrcd. Thk Inay have been due to exceptionally carI> dcgcncr~~t~on or lack of differentiation of the enamel epithelium in that area. Thus large deep pits in the enamel based b! cementum may have been enamel-fret originally. The problem of what prompts this physiological osteoclastic activity in the horse is puzzling. The larger areas of unrepaired resorption bays in the horse incisor compared with the molars may he related to a faster rate of formation of the incisors at ;I similar dcvelopmental stage. In the region of the ucll-marked perikymata of the molars. the sequence of developmental stages would be compressed into a more limited length of the tooth. The osteoclastic activity is brief: the pattern of bands and rings of resorption bays suggests the existence of waves of rcsorptibc actil‘ity moving 0ve1 the surface. lt is impossible to sa> from this stud) whether the rcsorptive cells themselves move on across the surface to fresh fields. or whether. after a brief activity. they die or change their function whilst a new lint of cells takes up the process of resorption. Osteociasts resorbing bones are very active mobile cells. Although they remain vital for only 2 or 3 dabs m tissue culture (Hancox. 1972). this may not ret&t their life irk I>~IYJ. The significance of the process of resorption of the enamel before cement deposition is casicr to guess. The surface area for the attachment of the coronal cetncnturn is increased in three ways: firstly because of the resorption bays, secondly by the roughness due to the different rate of etching of the prism hodie> and the inter-rowsheetinterprismatic arcas. and thirdly hecausc of the variation in the rate of etching of single crystals, or groups of crystals. which products a tint porosity ol the surface. Since the adhesion of the cementurn to enamel would be enhanced at all three levels of roughness bq the interlocking of the tissues. one wonders why such resorption is not encountered in other species. Of coronal cementogcncsis in the othet mammals which wc have invcstigatsd. that of the African elephant is the most like. A large bulk 01 vascular and cellular ccmentum is laid down rapidly on the enamel of the molars in a similar manner to the equine teeth but, although irregularities and dcficienties in the enamel surface were commonly found in the molars, resorption bays were not. The fine pattern of resorption been in the horse enamel is very similar to the etch pattern produced b> acids under laboratory conditions.Tuo levels of rctcntion only are obtained by such etching: firstly that due to the three-dimensional pattern produced by the var\ing orientation of the crystals in the prlsims and hence their different rates of solution. and secondly the finer pattern produced bt uneven etching rates of the crystals with similar orientation which products a porosity of significant depth (Ho~dc. Jonch and Reynolds. unpublished).
Coronal cementogenesis in the horse Resorption of the enamel surface in the horse does not follow immediately upon completion of the full thickness of the tissue. ft is probable, therefore, that the enamel has time partly to mature before resorption and cementum formation occur. Listgarten (1968) found that none of the enamel in the developing bovine molars he examined was fully mineralized before cementum was deposited on the surface. It is unlikely that the enamel could continue to mature beneath the cementurn because such maturation would involve not only the deposition of more mineral, but also the withdrawal of water and organic matrix. The X-ray emission micro-analysis data from the horse teeth conhrmed that no further maturation occurs after cementogenesis in the deciduous teeth, and suggested that the level of mineralization attained in the permanent incisor was initially considerably higher than that in the other teeth. It is debatable whether the degree of mineralization reached by the enamel affects either the rate or the extent of the subsequent resorption. The granular surface layer of equinine enamel could be a combination of the final secretion of the ameloblasts and organic matrix withdrawn from the immature enamel and then mineralized without being absorbed by the ameloblasts. This might explain the nodular. rough layer on the crests of the perikymata of some molars. Functionally. this might be significant in increasing the mechanical interlocking between enamel and cementum in any areas which are not resorbed prior to cementum deposition. The pattern of mineralization of the “intrinsic” matrix fibres of the cementum varies from one similar to that of primary bone (Fig. 18 : see also Boyde, 1972) to that of adult lamcllar bone (Figs. 19 and 20). We speculate that this is due to ditferences in the rate of formation and mineralization of the cementurn. +!,PUN /~,r/Hr,irc,,ll.s~~This work has been supported by the Medical Research Council and the Science Research Council. WC arc grateful to P. Reynolds and Elaine Bailey for technical assistance.
REFERENCES
Boydc A. 1964. The structure and development of mammalian enamel. Ph.D. Thesis, University of London. Boyde A. 1969. Correlation of ameloblast size with enamel prism pattern: use of scanning electron microscope to make surface area measurements. Z. Zellforsch. 93, 583593. Boy& A. 1970. The surface of enamel in human hypoplastic teeth. .4rchs orul Biol. 1.5, 897-898. Boyde A. 1972. Scanning electron microscope studies of hone In: Tin Biocherni.str.r und Physiology ofBone (edited by Bournc G. H.), 2nd Edn, Vol. I. Academic Press-New York.
609
Boyde A. and Jones S. J. 1972. Scanning electron microscope studies of the formation of mineralized tissues. In: Droe/oprttmtu~ Aspects ofOral Biology (edited by Slavkin H. C. and Bavetta L. A.). Academic Press, New York, Boyde A. and Wood C. 1969. Preparation of animal tissues for surface-scanning electron microscopy. J. Microsc. 90, 22 l--249. Franklin C. D. 1972. Ankylosis of an unerupted third molar by inostosis of enamel. Br. dmr. J. 133, 346-347. Glimcher M. J., Freiberg U. A. and Levine P. T. 1964. The identification and characterization of a calcified layer of coronal cementum in erupted bovine teeth. J. u/tr.trsttwt. Rcs. IO, 76-88. Hancox N. M. 1972. The ostcoclasts. In: 7‘hcBio~,/l[,/tri.r/,,l, md I’h\..\io/o~q,v of Bow (edited b! Bourns G. H.). 2nd Edn. Vol. 1. (‘hap. 3. Academic Press. New York. Havcrs C‘. 1691. 5th discourse on hones and tocth IO (he Royal Society (1689) In: Ostroiogiu Nocu 01 Soare ,%‘en Ohsrwutions of the Bones. Smith, London. Hunt A. M. 1959. A description of the molar teeth and investing tissues of normal guinea pigs. J. dent. Rex 38, 216231. Jones S. J. and Boyde A. 1972. A study of human root cementum surfaces as prepared for and examined in the scanning electron microscope. Z. Zr&r.xh. 130, 318338. Kawai N. 1955. Comparative anatomy of the bands of Schreger. Okujimas Fo/. mat. jap. 27, I 15-~13 1. Kronfeld R. 1938a. Coronal cementum and coronal resorption. J. dent. Rex 17, 151-159. Kronfeld R. 1938b. The biology of cementum. .I. 4~1. drrrf. Ass. 25, 1451~1461. Listgarten M. A. 1968. A light and electron microscopic study of coronal cementogenesis. Archs ortrl Biol. 13. 93114. Listgarten M. A. and Kamin A. 1969. The development of a cementurn layer over the enamel surface of rabbit molars-a light and electron microscopic study. Arch\ orul Biol. 14, 961-985. Longmore B. 1968. Coronal cementum in the rabbit. guinea pig and the horse. J. dent. Rrs. 47,997 -998. Mills P. B. and Irving J. T. 1967. Coronal cementogenesis in cattle, 4rch\ orrrl Rio/. 12, 929- 932. Neubauer C. and Schnitger A. 1970. Fin ncues Betrachtungsgerat fur Stereohildpaare aller Formrte. Bcitr. elektronen mikrosop. Direkruhh. Ohcr$. 3,41 I 41-I. Owen R. 1845. Odontograph~~. Balhere. London. Purkinje J. E. 1835. In: Fraenkel M. De Prnitori Drntiurn Hummorw~ Strucfurcr Oh.wrrc~tionrv. Inaug. Drss. Breslau. Rose C. 1894. iiber den Zahnbau und den Zahnwechsel von Elephas indicus. Molphol.
Schroeder
ofthe
4rh. 3, 173 194.
H. E. and Listgarten Dewloping
Epitheliul
M. A. 1971. Attuchrnent
FineSt,uctuw
of’ Humw~
7&h.
Karger, Base]. Schmidt W. J. and Keil A. 1971. Polurkin~g mi~w~.scvp!’ of Denrul Ti.ssw,s. Pergamon Press, Oxford. Wasserman B. H., Moskow B. S. and Rennert M. C. 1970. Dental anatomy and corona1 cementum m the Mongolian gerbil. J. pwiodont. Rex 5, 208-218. Weinreb M. M. and Sharav Y. 1964. Tooth development in sheep. 4rn. J. err Rm. 25, 89 l-908.
610
Sheila J. Jones and A. Boyde R&umC --La surface de I’~ma11. recouverte dc cement. ainsi que lc dt;\eloppemcnt ct la structure du cCment coronaire des dents de cheval sont btudits par microscopic Clectronique par balay,age. L.&mail c\t tout d’abord form6 de la mPme faqon que dans I’Cmail de mammif&e, chcL lcqucl 11 n’y a pas dc formation de cement. Cependant I’Cmail ne subit pas unc maturation cornpI&. SI I’on en Jugc par la concentration en calcium mesurCe par l‘analyse CltYmcntaire cn rayons X. Dc plus. une r?sorption partielle de la surface de I’t:mail prCctde la c6mcntogen&e. Le ci:mcnt c
Figs.
I-20
inclusive
and
Fig. 22 show neonate horse deciduous teeth: erupted teeth from a young adult horse.
Figures
21. 23 and
24 show
Fig. I. Tomes process pits at completion of the full enamel thickness with bumps and deprcsaions their floors and. to a lesser extent, on the ridges between the pits. Field width 20 Itm. Fig. 2. Pattern
I enamel pits in deciduous
molar with a circumferential of the pit. Field width I I llrn.
Fig. 3. Stereopair. Fig. 4. Pattern
Deciduous
II alignment
molar
ridge at the periphery
in
of the floor
pits with sloping floors m which the extra ridge has fused with the occlusal wall. Field width I I /rm_
of narrow.
deep pits. Field uidth
20 ,trn
Fig. 5. Surface enamel has blurred the crests of the inter-row sheets. Parts of three trcsorption left and early etching of the surface at lower ccntrc. Field width 50 ~cm.
bays arc at
Coronal cementogenesis
in the horse
PLATE I
A.O.B.
f.p. 610
SHEILA J.JONES and A. BOYDE
PLATE 2
Coronal
cementogenesis
in the horse
611
PLATE 2 Fig. 6. Rough globular
deposition on crest of perikymata above contrasts with the thin deposit enamel in the trough. Deciduous molar. Field width 100 pm.
Fig. 7. Pattern II enamel in the troughs and Pattern I enamel partly obscured deposit of surface enamel on the crests of the perikymata of a deciduous molar. Fig. 8. Large hypoplastic Fig. 9. Circular
resorption
defects in the enamel at approximately Field width 500 itm. bay in enamel
surface.
Parts
Fig. 10. Resorbing areas on molar enamel. Small isolated is ridged longitudinally, as well as horizontally Fig.
11. Island of unresorbed
enamel
by granular, globular Field width 240 pm.
the same developmental
of two others
of surface
level in a molar
at top right. Field width 50 pm.
pits can be seen in lower part. The molar surface by the perikymata. Field width 1 mm.
ringed by resorption bays in area specimen. Field width 745 pm.
of cementogenesis.
Anorganic
Sheila J. Jones and A. Boyde
Fig. 12. Junction 01‘ resorbed cnCimcl (ahove) and cemcntogenesls (hrloti 1. The pattern of the cnamcl ia shov,n 111the 1x1~ due tu the dill’crcncc m the etch rates 01 the prism boundary and ccntrc. The stcrcopair illustrates
the depth
of the
prismatic
resorption
hays.
viewer (Neuhauer
(This
pair
and Schnitger.
should
he viewed
with
1970).) Field width
a System
46 I’m.
Nesh
type
Coronal cementogenesis
in the horse
PLA
.TE 3
A .O.B. f.p. 612
SHEILA J. JONES
PLATE 4
and
A. BOYD~
Coronal
cementogenesis
in the horse
613
PI A,,. 4 Fig. 13. Rough,
irregular
Fig. 14. Three orders
surface produced by resorption of enamel. Prism different rates of resorption. Field width 100 pm.
of roughness:
due to the bays. the prisms and the crystals.
structure
emphasized
by
Field width 20 pm.
Fig. IS. Narrow advancing edge of resorption with bays with their long axes at right angles to the direction of advance. Cementogenesis is repairing the resorption in right half of the picture. Unresorbed enamel at left. Anorganic specimen. Field width 240 pm. Fig. 16. Randomly Fig. 11.Random Fig. 18. Pattern
arranged
collagen
collagen
fibres in the plane of the forming 59 /Irn.
fibrils in back wall of half-formed
cementum
lacuna in cementurn
surface.
Field width
surface. Field width 24 ,um.
of mineralization of cementum with small mineral particle aggregates. at centre. Anorganic specimen. Field width 55 pm.
Cementocyte
lacuna
hl4
Sheila J. Jones and A. Boyde
t tg. 19. Later stage of mineralization Three cementocyte lacunae Fig. 20. Centre
lacuna
with larger spindle-shaped mineralized at centre horizon. Anorganic specimen,
of Fig. 19. showing
fine fibriikdr meshwork width 24 pm.
segments of collagen Field width 72 pm.
of fully mineralized
tibres.
back waif. Fiefd
Fig. 21. Completion of cementogencsis: the openings of the canaliculi are obvious in the fully mineralized surface. Cementocyte lacuna at centre is bordered by two Sharpey fibres which are not completely mineralized. Anorganic specimen. Field width 25 pm. k’ig. 22. Clefts or pits remain
in the cementum where resorption of the enamel Occlusal to bottom right. Field width 5 mm.
has not yet occurred.
Fig. 23. Surface of erupted molar: scratches due to abrasion cover the cementum. Vascular openings are regularly placed at the bases of hollows. Many cementocyte lacunae are exposed at the surface. Field width 420 pm. Fig. 24. Fractured anorgamc ccmentum. The intrinsic tibres are not well mineralized, and the Sharpey tibres running from top left to bottom right have unmineralized cores and a constricted pattern of fibre mineralization. Field width 50 pm.
Coronal
cementogenesis
in the horse
PLATE 5 A.O.R. f.p. 614