Archs oral Bid.
Vol.
20. pp. 551 to 552. Pergamon
MEMBRANE
Press. Printed
m Great
Braan.
JUNCTIONS G. R.
ON CAT ODONTOBLASTS HOLLAND
Department Anatomy (Oral Biology), University of Bristol, Medical School. University Walk. Bristol, BS8 ITD, England
Summary--The odontoblast layer of the pulp was examined after perfusion fixation with buffered aldehydes. Desmosomal junctions occur between only some of the odontoblasts at the predentina.1 border. Pale cytoplasmic processes similar in morphology to nerve terminals in the intercellular spaces make specialized junctions on the cell bodies of the odontoblasts which may be small subunit gap-junctions of low electrical resistance.
Ultrastructural observations on the human odontoblast layer have established the existence of various intercellular junctions. At the predentine surface, junctional complexes between adjacent odontoblasts were described by Frank (1968) and Furseth (1971). At a deeper level, Frank (1966) and Arwill (1968) found desmosomes uniting the odontoblast cell bodies, which, over the greater part of their surface, were separated from one another by large intercellular spaces. Frank (1966, 1968) and Dahl and Mjor (1973) found narrow cytoplasmic processes in these spaces and suggested that some of them were nerve fibres. Dahl and Mjor reported occasional tight junctions between the:;e cytoplasmic processes and the odontoblast cell bodies; some actually invaginated into the odontoblast. In connection with electrophysiological studies of receptor mechanisms in the teeth of cats, Frank (1972) turned his attention to the ultrastructure of dentine and pulp in these animals. He found pale cytoplasmic processes lying bel:ween the odontoblast cell bodies, but did not describe any special relationship between the processes and the odontoblasts; he considered some of these processes to be nerve fibres. The present paper reports a preliminary investigation on the odontoblast cell body layer, to eiucidate inter-odontoblast relationships and possible connections between the odontoblasts and the pale cytoplasmic processes described by Frank. Young adult cat:; anaesthetized with sodium pentobarbitone were perfused for 2G30 min with a mixture of aldehydes (1 g/l 00 ml each of glutaraldehyde, formaldehyde and acrolein) in Tyrode’s solution. The osmotic pressure of the mixture was 630 milliosmoles and of the Tyrode’s solution 288 milliosmoles, as measured by the freezing point depression method. After the perfusiojn, the canine teeth were dissected out and slit transversely under a continuous spray of Tyrode’s solution using a diamond disc mounted in a lathe. The tissue blocks obtained as transverse sections were post-,fixed in 1 per cent osmium tetroxide and embedded in Araldite. After removal of the hard tissue, ultrathin sections were cut from the odontoblast layer. These were double-stained with uranyl acetate and lead citrate, examined and photographed in a Philips 300 electron microscope. At the pulpal margin of the predentine (or the predentine surface 551
of the pulp), the odontoblasts lay in close contact with each other (Fig. 1) but there were no examples of complete junctional complexes. Occasional desmosomes were seen (Fig. 3). Large intercellular spaces were present between adjacent cell bodies deep to the predentine surface (Fig. 2) similar to the appearance described by Arwill (1967, 1968). The odontoblasts were arranged around these spaces in a stellate pattern, with projecting arms of cytoplasm making contact with similar projections from neighbouring cells. Occasionally there were desmosomes between contacting cytoplasmic projections. but generally the contacts showed no specialization. Islands of cytoplasm (Fig. 2) paler than the odontoblast cytoplasm were visible in the intercellular spaces which otherwise seemed to be empty except for a patchy granular background. These cytoplasmic islands contained microtubules, microfilaments and often also mitochondria. They frequently established specialized connections with the odontoblast cell bodies (Fig. 4). Generally the connections were near the point at which the two cell bodies met (Fig. 2). These junctions resembled the occludens type as described by Farquar and Palade (1963) in epithelia. the outer leaflet of the apposed plasmalemmas being fused to form a 5-layered structure. However, McNutt and Weinstein (1973) point out that small sub-unit gap-junctions can often resemble occludens junctions, and they suggest that many junctions described in the past as tight junctions are really gap junctions. In the present study, measurements of the thickness of five of the junctions between the cytoplasmic islands and the odontoblasts showed them to be never less than twice the thickness of neighbouring nonjunctional plasma membrane. According to McNutt and Weinstein, this would put them in the category of small subunit gap-junctions rather than the occludens type. The intercellular spaces in this study and that of Arwill (1967, 1968) are larger than those shown by other workers (Johannsen, 1967; Frank, 1966; Nalbandian, 1968). Their size in fixed tissue depends on the tonicity of the fixative used. Recent studies (Bone and Ryan. 1973), together with light microscopic studies (Young, 1935), suggest that it is the osmolarity of the buffer vehicle rather than the osmolarity of the whole fixation fluid that is important in control-
Membrane junctions
Fig. 1. The pulp-predentine
junction.
on cat odontoblasts
PD =predentine.
D=desmosome.
x
20,000
Fig ;. 2. The odontoblast cell bodies at the level of their nuclei. Arrows indicate specialized junctions between pale cytoplasmic processes and odontoblast cell bodies. x 13,000 Fig :. 3. A desmosome
linking two odontoblasts at the predentinal x 70,000
border. Enlarged
Fig ;. 4. A nerve-like process making a specialized junction with an odontoblast
from Fig. 1.
cell body.
x
104,000 A.O.B.
f.p. 552
552
G. R. Holland
ling cell volume in the fixed tissue. On this basis, the fixative used in this investigation should cause minimal shrinkage. The junctions between odontoblasts at the predentine margin differ from those in man. As these junctions may control the access of chemicals to the pulp. this difference may help to explain the apparently different responses of human and cat dentine to some chemical stimuli (Horiuchi and Matthews. 1972). Although many of the pale cytoplasmic processes in the intercellular spaces are consistent with the criteria Frank (1966) used to recognise intratubular nerve fibres, their nature cannot be deduced solely from their appearance. However. the possiblity that these nerve-like structures are in contact with odontoblasts by gap-junctions is interesting. Gap-junctions provide a low electrical resistance pathway between cells (Pappas, Asada and Bennet, 1971) and may therefore provide a pathway of low electrical resistance between odontoblasts and nerve fibres. More numerous measurements after standardized preparatory procedures together with studies using markers of extracellular space such as lanthanam hydroxide are needed to establish these as gap-junctions. Ultrastructural histochemistry and nerve section experiments are necessary to determine which if any of the structures forming the junctions are neural. Serial reconstructions would show the precise anatomical relations of the odontoblast in this region but none of these measures would establish the odontoblast as a receptor. It may support the nerve fibre metabolically or transmit mechanical disturbance. The excitability of the odontoblast can only be demonstrated by precise intracellular electrophysiological techniques. Ackrtowlrdgernrnts~This work was supported by a grant from the Medical Research Council. I am grateful to Mr. I. P. Rogers for his adept technical assistance and to Miss Susan Jones for photographic help.
REFERENCES
Arwill T. 1967. Studies on the ultrastructure of dental tissues-11: The predentine-pulpal border zone. Odont. Rec. 18, 191-308. Arwill T. 1968. The ultrastructure of the pulpo-dentinal border zone. In: Drrltirlr und Pulp (Edited by Symonds N. B. B.) p. 147~168. Livingstone. London. Bone Q, and Ryan K. P. 1972. Osmolarity of osmium tetroxide and glutaraldehyde fixatives. Histochern. J. 4.331-347. Dahl E. and Mjor I. A. 1973. The distribution of nerves in the pulp-dentine organ. Acta odorlt. scmd. 31. 34% 356. Farquar M. G. and Palade G. E. 1963. Junctional complexes in various epithelia. J. cell Biol. 17. 37S412. Frank R. M. 1966. Etude au microscope Clectronique de I’odontoblast et due canalicule dentinaire humain. Archs ora/ Biol. 11, 179.-199. Frank R. M. 1968. Ultrastructural relationship between the odontoblast. its process and the nerve fibre. In: Drntiw arId Pulp (Edited by Symonds N. B. B.) pp. 115~145. Livingstone. London. Furseth R. 1971. Tight junctions between osteocyte processes. &and. J. dent. Rrs. 81, 339-341. Horiuchi H. and Matthews B. 1972. Neural responses evoked by stimulation of dentine. In: Oral Physiology (Edited by Emmelin N. and Zotterman Y.). Pergamon, Oxford. Johannsen R. 1957. Ultrastructure of dentine. In: Structural and Chmicul Orgunisatiou of Teeth (Edited by Miles A. E. W.), Vol. II, pp. 35-76. Academic Press, London. McNutt N. A. and Weinstrin R. S. 1973. Membrane ultrastructure at mammalian intercellular junctions. Prog. Biophys. mol. Biol. 26. 45p IO I. Nalbandian J. 1968. The ultrastructure of odontoblasts and dentinogenesis. In: Biology qf the Dental Pulp Organ (Edited by Finn S. B.). University of Alabama Press, Alabama. Pappas G. D.. Asada Y. and Bennet M. V. L. 1971. Morphological correlates of increased coupling resistance at an lectronic synapse. J. cell Biol. 49. 173%188. Young J. L. (1935) Osmotic pressure of fixing solutions. Nuture (Lond.) 135. 823~-829.
Vol.
20. pp. 551 to 552. Pergamon
MEMBRANE
Press. Printed
m Great
Braan.
JUNCTIONS G. R.
ON CAT ODONTOBLASTS HOLLAND
Department Anatomy (Oral Biology), University of Bristol, Medical School. University Walk. Bristol, BS8 ITD, England
Summary--The odontoblast layer of the pulp was examined after perfusion fixation with buffered aldehydes. Desmosomal junctions occur between only some of the odontoblasts at the predentina.1 border. Pale cytoplasmic processes similar in morphology to nerve terminals in the intercellular spaces make specialized junctions on the cell bodies of the odontoblasts which may be small subunit gap-junctions of low electrical resistance.
Ultrastructural observations on the human odontoblast layer have established the existence of various intercellular junctions. At the predentine surface, junctional complexes between adjacent odontoblasts were described by Frank (1968) and Furseth (1971). At a deeper level, Frank (1966) and Arwill (1968) found desmosomes uniting the odontoblast cell bodies, which, over the greater part of their surface, were separated from one another by large intercellular spaces. Frank (1966, 1968) and Dahl and Mjor (1973) found narrow cytoplasmic processes in these spaces and suggested that some of them were nerve fibres. Dahl and Mjor reported occasional tight junctions between the:;e cytoplasmic processes and the odontoblast cell bodies; some actually invaginated into the odontoblast. In connection with electrophysiological studies of receptor mechanisms in the teeth of cats, Frank (1972) turned his attention to the ultrastructure of dentine and pulp in these animals. He found pale cytoplasmic processes lying bel:ween the odontoblast cell bodies, but did not describe any special relationship between the processes and the odontoblasts; he considered some of these processes to be nerve fibres. The present paper reports a preliminary investigation on the odontoblast cell body layer, to eiucidate inter-odontoblast relationships and possible connections between the odontoblasts and the pale cytoplasmic processes described by Frank. Young adult cat:; anaesthetized with sodium pentobarbitone were perfused for 2G30 min with a mixture of aldehydes (1 g/l 00 ml each of glutaraldehyde, formaldehyde and acrolein) in Tyrode’s solution. The osmotic pressure of the mixture was 630 milliosmoles and of the Tyrode’s solution 288 milliosmoles, as measured by the freezing point depression method. After the perfusiojn, the canine teeth were dissected out and slit transversely under a continuous spray of Tyrode’s solution using a diamond disc mounted in a lathe. The tissue blocks obtained as transverse sections were post-,fixed in 1 per cent osmium tetroxide and embedded in Araldite. After removal of the hard tissue, ultrathin sections were cut from the odontoblast layer. These were double-stained with uranyl acetate and lead citrate, examined and photographed in a Philips 300 electron microscope. At the pulpal margin of the predentine (or the predentine surface 551
of the pulp), the odontoblasts lay in close contact with each other (Fig. 1) but there were no examples of complete junctional complexes. Occasional desmosomes were seen (Fig. 3). Large intercellular spaces were present between adjacent cell bodies deep to the predentine surface (Fig. 2) similar to the appearance described by Arwill (1967, 1968). The odontoblasts were arranged around these spaces in a stellate pattern, with projecting arms of cytoplasm making contact with similar projections from neighbouring cells. Occasionally there were desmosomes between contacting cytoplasmic projections. but generally the contacts showed no specialization. Islands of cytoplasm (Fig. 2) paler than the odontoblast cytoplasm were visible in the intercellular spaces which otherwise seemed to be empty except for a patchy granular background. These cytoplasmic islands contained microtubules, microfilaments and often also mitochondria. They frequently established specialized connections with the odontoblast cell bodies (Fig. 4). Generally the connections were near the point at which the two cell bodies met (Fig. 2). These junctions resembled the occludens type as described by Farquar and Palade (1963) in epithelia. the outer leaflet of the apposed plasmalemmas being fused to form a 5-layered structure. However, McNutt and Weinstein (1973) point out that small sub-unit gap-junctions can often resemble occludens junctions, and they suggest that many junctions described in the past as tight junctions are really gap junctions. In the present study, measurements of the thickness of five of the junctions between the cytoplasmic islands and the odontoblasts showed them to be never less than twice the thickness of neighbouring nonjunctional plasma membrane. According to McNutt and Weinstein, this would put them in the category of small subunit gap-junctions rather than the occludens type. The intercellular spaces in this study and that of Arwill (1967, 1968) are larger than those shown by other workers (Johannsen, 1967; Frank, 1966; Nalbandian, 1968). Their size in fixed tissue depends on the tonicity of the fixative used. Recent studies (Bone and Ryan. 1973), together with light microscopic studies (Young, 1935), suggest that it is the osmolarity of the buffer vehicle rather than the osmolarity of the whole fixation fluid that is important in control-
Membrane junctions
Fig. 1. The pulp-predentine
junction.
on cat odontoblasts
PD =predentine.
D=desmosome.
x
20,000
Fig ;. 2. The odontoblast cell bodies at the level of their nuclei. Arrows indicate specialized junctions between pale cytoplasmic processes and odontoblast cell bodies. x 13,000 Fig :. 3. A desmosome
linking two odontoblasts at the predentinal x 70,000
border. Enlarged
Fig ;. 4. A nerve-like process making a specialized junction with an odontoblast
from Fig. 1.
cell body.
x
104,000 A.O.B.
f.p. 552
552
G. R. Holland
ling cell volume in the fixed tissue. On this basis, the fixative used in this investigation should cause minimal shrinkage. The junctions between odontoblasts at the predentine margin differ from those in man. As these junctions may control the access of chemicals to the pulp. this difference may help to explain the apparently different responses of human and cat dentine to some chemical stimuli (Horiuchi and Matthews. 1972). Although many of the pale cytoplasmic processes in the intercellular spaces are consistent with the criteria Frank (1966) used to recognise intratubular nerve fibres, their nature cannot be deduced solely from their appearance. However. the possiblity that these nerve-like structures are in contact with odontoblasts by gap-junctions is interesting. Gap-junctions provide a low electrical resistance pathway between cells (Pappas, Asada and Bennet, 1971) and may therefore provide a pathway of low electrical resistance between odontoblasts and nerve fibres. More numerous measurements after standardized preparatory procedures together with studies using markers of extracellular space such as lanthanam hydroxide are needed to establish these as gap-junctions. Ultrastructural histochemistry and nerve section experiments are necessary to determine which if any of the structures forming the junctions are neural. Serial reconstructions would show the precise anatomical relations of the odontoblast in this region but none of these measures would establish the odontoblast as a receptor. It may support the nerve fibre metabolically or transmit mechanical disturbance. The excitability of the odontoblast can only be demonstrated by precise intracellular electrophysiological techniques. Ackrtowlrdgernrnts~This work was supported by a grant from the Medical Research Council. I am grateful to Mr. I. P. Rogers for his adept technical assistance and to Miss Susan Jones for photographic help.
REFERENCES
Arwill T. 1967. Studies on the ultrastructure of dental tissues-11: The predentine-pulpal border zone. Odont. Rec. 18, 191-308. Arwill T. 1968. The ultrastructure of the pulpo-dentinal border zone. In: Drrltirlr und Pulp (Edited by Symonds N. B. B.) p. 147~168. Livingstone. London. Bone Q, and Ryan K. P. 1972. Osmolarity of osmium tetroxide and glutaraldehyde fixatives. Histochern. J. 4.331-347. Dahl E. and Mjor I. A. 1973. The distribution of nerves in the pulp-dentine organ. Acta odorlt. scmd. 31. 34% 356. Farquar M. G. and Palade G. E. 1963. Junctional complexes in various epithelia. J. cell Biol. 17. 37S412. Frank R. M. 1966. Etude au microscope Clectronique de I’odontoblast et due canalicule dentinaire humain. Archs ora/ Biol. 11, 179.-199. Frank R. M. 1968. Ultrastructural relationship between the odontoblast. its process and the nerve fibre. In: Drntiw arId Pulp (Edited by Symonds N. B. B.) pp. 115~145. Livingstone. London. Furseth R. 1971. Tight junctions between osteocyte processes. &and. J. dent. Rrs. 81, 339-341. Horiuchi H. and Matthews B. 1972. Neural responses evoked by stimulation of dentine. In: Oral Physiology (Edited by Emmelin N. and Zotterman Y.). Pergamon, Oxford. Johannsen R. 1957. Ultrastructure of dentine. In: Structural and Chmicul Orgunisatiou of Teeth (Edited by Miles A. E. W.), Vol. II, pp. 35-76. Academic Press, London. McNutt N. A. and Weinstrin R. S. 1973. Membrane ultrastructure at mammalian intercellular junctions. Prog. Biophys. mol. Biol. 26. 45p IO I. Nalbandian J. 1968. The ultrastructure of odontoblasts and dentinogenesis. In: Biology qf the Dental Pulp Organ (Edited by Finn S. B.). University of Alabama Press, Alabama. Pappas G. D.. Asada Y. and Bennet M. V. L. 1971. Morphological correlates of increased coupling resistance at an lectronic synapse. J. cell Biol. 49. 173%188. Young J. L. (1935) Osmotic pressure of fixing solutions. Nuture (Lond.) 135. 823~-829.
