Archs oral Bid. Vol. 36, No. 9, pp. 641646, 1991 Printed in Great Britain. All rights reserved
0003-9969/9153.00+ 0.00 Copyright 0 1991Pergamon Press plc
THE PERMEABILITY OF CAT DENTINE AND IN VITRO
IN VW0
N. VONGSAVANand B. MATTHEWS Department of Physiology, University of Bristol, University Walk, Bristol BS8 ITD, U.K. (Accepted 19 March 1991) Summary-The apparent impermeability of dentinal tubules to chemicals applied to exposed dentine in vivo was investigated. It was shown that whereas Evans’ blue diffused readily into dentine in extracted teeth, it did not do so in visible amounts when it was applied in vim. In the invitroexperiments, decreasing the pressure of the Evans’ blue solution to 20 cmH,O below atmospheric apparently prevented the dye entering the tubules, while increasing the pressure of the solution to 15 cmH,O or more above atmospheric in vivoresulted in the dye penetrating the dentine in high concentrations. It is concluded that, in cat dentine in vim, there is an outward flow of fluid through exposed dentinal tubules, and that this flow is sufhcient to substantially reduce diffusion into the tubules of chemicals such as Evans’ blue. Key words: dentine permeability
INTIIODUCTION Anderson
and
Ronning
(1962) demonstrated
that
human dentine is freely permeable to the dye Evans’ blue. They made their observations on recently extracted teeth, and on dentine that had been exposed by fracturing off the tips of cusps. Several observations in this laboratory have suggested that cat dentine in vivo may not be as permeable as indicated by these experiments. F:or example, when solutions of cobalt chloride were applied to exposed dentine in vivo, no evidence could be found that cobalt ions diffused into the dentinal tubules or pulp (Matthews, 1976). The dentine was exposed by fracturing off approx. 1 mm from th.e tip of the cusp of a canine tooth under Ringer’s solution. This procedure was used to avoid the tubules becoming obstructed by air being sucked into them, by debris from a drill, or by a smear layer (Pashley et al., 1988). A similar result was obtained in preliminary experiments with solutions of Evans’ blue and horseradish peroxidase, and the result with the peroxidase has recently been confumed at the ultrastructural level (Matthews B. and Hughes S. H. S., unpublished observations). Further evidence that there is greater resistance to inward diffusion of substances through dentine in vivo than would be. expected from the studies on extracted teeth has been obtained in experiments on cats in which it was found that topical applications of local anaesthetic solutions or fixatives did not abolish neural responses evok’ed by mechanical stimulation of the dentine. The dentine was exposed as described above, but using a diamond disc rather than fracturing, and it was etched with acid to remove the smear layer. The only evidence that substances can diffuse through dentine in vivo has been obtained using radio-isotopes of iodine in cats (Edwall and Kindlova, 1971) and dogs (Pashley et al., 1981a) and in these instances the smear layer was not removed.
A smear layer would also have been present in the experiments of Horiuchi and Matthews (1974) in which they found that topical applications of 2% lignocaine to exposed dentine in cat teeth had no effect on the propagation of impulses in pulpal nerves. We have now investigated further the permeability of cat dentine in vitro and in vivo. MATERIALS AND
METHODS
All the observations were made on the permanent canine teeth of young adult cats. The cats were anaesthetized with sodium pentobarbitone (Sagatal; May and Baker, Dagenham, U.K. 42mg/kg intraperitoneally followed by 3 mg/kg intravenously as required). The permeability of the coronal dentine of these teeth to the dye Evans’ blue was investigated under three conditions: in teeth which had been extracted, in teeth which remained in situ with an intact blood supply, and in teeth which remained in situ but with their root pulps sectioned.
The teeth that were ex-
tracted before testing were removed surgically with minimal force. They were stored in Ringer’s solution and the dentine was exposed immediately before its permeability was investigated. Care was taken to prevent the dentine drying. In those teeth in which the pulp was sectioned, this was done in the middle third of the root after the pulp had been exposed by removing the overlying bone and dentine. There is no evidence that blood vessels enter the pulp of the cat canine other than in the apical region of the root and therefore this procedure will have stopped all blood flow to the coronal pulp. With both groups of teeth that were tested in situ, a section of dentine was removed for histological examination immediately after the period of dye application and without extracting the teeth.
641
N.
642
VONGSAVANand
B.
MATTHEWS
Further details of the methods used in individual experiments are given at the appropriate points in the Results section.
Collar
Evans’
Blue
RESULTS
Dentine exposed by fracturing
Tooth
Per-ipex
Cop
4 mm
I
Fig. 1. Scale diagram of the cap used to apply Evans’ blue to exposed dentine of cat canine teeth in vitro and in vivo.
Dentine exposed by fracturing Apart from the method of exposing the dentine, the basic preparation of the tooth was the same for all the experiments (Text Fig. 1). The surface of the enamel was cleaned and etched with 4.6 mol/l orthophosphoric acid for 30 s. Then a Perspex collar was fitted around the crown, 2-3 mm below the tip of the cusp, and sealed in place with resin (Concisea Enamel Bond System, 3M Dental Products, St Paul, MN, U.S.A.). The enamel was scored approx. 1 mm from the cusp tip and this part of the crown was fractured off with shears under Ringer’s solution. Approximately 1 mm of dentine remained over the pulp cornu. A small Perspex cap was sealed on to the collar to enclose the tip of the tooth, and filled with a solution of Evans’ blue (52 mmol/l) in isotonic saline. After 30 min, the Evans’ blue was washed out of the cap, the cap was removed, and an axial longitudinal section, approx. 0.5mm thick, was cut through the crown with a diamond disc under Ringers. The section was dehydrated overnight in 100% ethyl alcohol, cleared in methyl salicylate and examined under a microscope (Wild, type M4~) with variable dark-ground illumination. Evans’ blue is very much less soluble in ethyl alcohol or methyl salicylate than in water and therefore its diffusion through the dentine was effectively arrested when the section was placed in the alcohol. Dentine exposed with a diamond disc In some cases, both in vivo and in vitro, the same amount of tooth substance was removed from the tip of the tooth but instead of by fracturing it was removed with a diamond disc (Komet, Gebr. Brasseler, Lemgo, Germany. Type 943, thickness 0.15 mm) under a stream of Ringer’s solution. The exposed dentine surface was etched with either 4.6 mol/l ortho-phosphoric acid for 30 s or 0.1 moljl EDTA @H 7.0) for 5 min.
When Evans’ blue was applied for 30min to exposed dentine in extracted teeth (n = IO), it diffused through the opened tubules into the pulp and stained these tissues heavily [Plate Fig. 2(A)]. The teeth were examined at room temperature from 20 min to more than 10 h after extraction, having been stored in Ringer’s either at room temperature (n = 9) or frozen (n = I), and the result was the same in all cases. When the same procedures were carried out in vivo, no Evans’ blue could be detected in the dentinal tubules or pulp of the cat teeth (n = 9) [Plate Fig. 2(B)]. The most likely explanation seemed to be that there was a constant outward flow of fluid through the dentinal tubules in vim, caused by the pulpal tissuefluid pressure being above atmospheric; and that the rate of flow of this fluid was sufl’icient to severely reduce the rate of inward diffusion of the dye. We attempted to test this hypothesis by determining the effect on dye diffusion of cutting through the dental pulp near the root apex, thus arresting the flow of blood through the pulpal circulation of a tooth that was left in situ. In four teeth tested 1.5 h after cutting the root pulp, no dye could be detected in the dentine or pulp of three, but the dye reached the pulp in the other. The one tooth tested 10 h after pulp section was impermeable to the dye. We therefore concluded that fluid flowing through the dentinal tubules was probably not responsible for the lack of detectable dye penetration into the dentine. Other work in our laboratory (Matthews and Hughes, 1988) has demonstrated that the outer ends of the dentinal tubules over the pulp cornua in cat canines are not empty but contain a material that is more electron-dense than extracellular fluid when observed in stained sections in the transmission electron microscope. Similar observations have been made by others on specimens of both cat (Holland, 1975, 1985) and human (Tsatas and Frank, 1972; Thomas, 1979; Thomas and Payne, 1983) dentine. We next considered the possibility that this material might be acting as a plug, forming a barrier to diffusion; although it was not clear how it could become leaky soon after extraction of a tooth, and not do so in a tooth left in situ with its pulp cut.
Plate 1 Fig. 2. Photomicrographs of ground longitudinal sections of the crowns of cat canine teeth. The sections were dehydrated and cleared in methyl salicylate. In each case, a solution of Evans’ blue was applied for 30 min to the exposed dentine at the tip of the cusp immediately before the section was cut. The dye was applied under different conditions in each case. Original magnification: x 35. (A) Recently extracted tooth. (B) Dye applied in uivo, before the tooth was extracted. (C) Recently extracted tooth. The pressure in the cap containing the dye was maintained at ZOcmH,O below atmospheric throughout the period of dye application. (D) Dye applied in t&o. The pressure in the cap was maintained at 20cmHi0 above atmospheric.
Permeability of dentine
Plate 1
643
644
N. VONGSAVAN and B. MATTHEWS
Autolysis would be expected to proceed similarly under both conditions. Maybe the act of extraction, despite our care to minimize the trauma to the tooth, was causing the plugs to move in the tubules and to become leaky, or perhaps the temperature of the tooth during the period between the arrest of the circulation and testing was an important factor. The latter possibility was tested first. Extracted teeth were stored at either 4 (n = 6) or 37°C (n = 4) for periods from 20 min to 10 h before the permeability of the dentine was tested in vitro. All these teeth were found to be permeable to Evans’ blue. Some teeth were also left in situ with their pulps intact and tested after death of the animal. The body was stored at room temperature until the teeth were tested. The dye penetrated the dentine 1.5 h after death (n = 2), but none was detectable 12 h after (n = 3). Thus while the results obtained from teeth both in vivo and in vitro after extraction were very reproducible, those obtained from teeth in situ after arrest of pulpal blood flow were very variable and did not fit into any obvious pattern. The only ways in which we could imagine that the surgical extraction of a tooth might dislodge plugs in the dentinal tubules over the pulp cornua were either if negative pressures were developed in the pulp during the extraction or if tension was applied to the odontoblast processes via connective tissue, nerves and vessels that passed through the apical foramen. Both mechanisms seemed improbable but were tested by opening the pulp cavity in the root and cutting through the pulp before the tooth was extracted. The dentine was permeable to Evans’ blue in all cases (n = 3), despite these precautions. We next considered the possibility that changes in the surface charge on the lining of the dentinal tubules was affecting their permeability to the dye. Evans’ blue is an acid dye (mol. wt 960) and therefore a change from a negative to a positive surface charge would tend to impede its penetration. This was tested by using the basic dyes Janus green (mol. wt 483) and methylene green (mol. wt 364). Methylene green has the advantage that, like Evans’ blue, it is relatively insoluble in alcohol. Neither Janus green (n = 1) nor methylene green (n = 4) penetrated dentine in vivo, but both dyes reached the pulp in vitro (n = 1 in both cases). Thus the surface charge on the inner surface of the tubules was not responsible for the differences observed. We finally returned to our original, fluid-flow hypothesis. Our test of this hypothesis would not have been valid if, for some reason, a significant pressure tending to force fluid out through the dentine remained in the coronal pulp after cutting the root pulp, but not after the extraction of a tooth. One possible mechanism which was considered was that at the time of pulp section in the root (but not for some reason after extraction), blood vessels there were occluded by vasoconstruction and clot formation, and that as a result pressure was maintained for some time in the vessels in the coronal pulp on account of its low compliance (Heyeraas K. J., 1990, personal communication). This possibility seems to have been eliminated, however, by the observation that dye
entered the dentine of extracted teeth in which the root pulp had been sectioned just before extraction (see above). An alternative possibility was that the pressure in the coronal pulp of a tooth in situ was maintained after section of the root pulp as a result of communication between the tissue fluid in the pulp and that in the periodontal tissues (in which the blood flow was largely intact) through the dentinal tubules and cementum of the root. The fluid-flow hypothesis was therefore tested in an alternative way, by increasing the hydrostatic pressure of the Evans’ blue in vivo and by decreasing it below atmospheric in vitro. When the pressure in the cap was reduced to 15 cmH,O below atmospheric in vitro, visible amounts of the dye still entered dentine (n = 2); but no dye was detectable after exposure at a pressure of -20 cmH,O (n = 6) [Plate Fig. 2(C)]. When the cap pressure was increased to lOcmH,O above atmospheric in vivo, Evans’ blue was not detectable in the dentine (n = 2), but it was visible with pressures of 15 and 20 cmH,O (n = 7 and 2, respectively) [Plate Fig. 2(D)]. Dentine exposed with a diamond disc
When dentine was tested after it had been exposed in vitro by cutting with a diamond disc instead of by
fracturing, no Evans’ blue could be detected in the tubules (n = 2). However, when the exposed dentine was etched with orthophosphoric acid or EDTA before testing, the dye entered the tubules and stained the pulp in all six teeth examined. These teeth were examined at room temperature and between 20min and 24 h after extraction. Dentine tested in vivo after it had been exposed with a diamond disc and etched with orthophosphotic acid, contained no detectable Evans’ blue (n = 3). The same result was obtained in one tooth that was etched with EDTA. DlSCUSSlON
Our observations on the permeability of dentine to Evans’ blue in recently extracted cat teeth agree with previous findings on human teeth (Anderson and Ronning, 1962). Under these conditions, as long as the tubules were not obstructed by a smear layer, Evans’ blue diffused rapidly through the dentine. When Evans’ blue was similarly applied to exposed dentine in vivo, none appeared to enter the dentine. This same result was obtained whether the dentine had been exposed by fracturing or by using a diamond disc, provided that the smear layer left by the disc was first removed by etching. The results obtained by changing the pressure gradient across the dentine, both in vitro and in vivo, indicated that the rate of outward flow of fluid through dentinal tubules determined the rate at which dye was able to diffuse upstream into them. Thus, we conclude that when dentine is exposed in vivo, there is an outward flow of fluid through the tubules and that this flow is sufficiently rapid to severely limit the rate of inward diffusion of chemicals. After extraction of a tooth, the pulpal tissue-fluid pressure falls and under these
Permeability of dentine conditions the rate of outward flow of fluid through the tubules decreases to the point that it no longer affects the rate of inward diffusion of the dye. In all of these. studies, the Evans blue solution would have added an osmotic effect, which would have tended to increase the outward flow of fluid. This is unlikely to have influenced the results significantly, however, in view of the low molar concentration of the dye and the fact that even in vivo it was shown that the dye was not prevented from entering the tubules by virtue of the size of its molecule. All of our findings can be explained on the basis of such a mechanism except those obtained from teeth left in situ but with no pulpal blood flow. The only explanation we can offer for the differences between these results and those obtained from extracted teeth is that, after section of the pulp of a tooth left in situ, the tissue-fluid pressure in the coronal pulp tended to be maintained at a hi,gher level than in extracted teeth as a result of communication with the tissue fluid of the periodontal ligam.ent through the dentinal tubules of the upper part of the tooth. Our findings also explain earlier ones which suggested that cat dentine in vivo was impermeable to substances such as horseradish peroxidase and local anaesthetics (see Introduction). They also account for other observations, including the following. It has been shown that nerve endings in teeth are not excited when chemical stimuli are applied to dentine unless only a thin layer of dentine remains over the pulp (Horiuchi and Matthews, 1976; NIhri and Hirvonen, 1987). Iwaku et al. (1981) reported that resin penetrated the ends of tubules more deeply when it was applied to etched dentine in vitro than in vivo. There is evidence that under certain conditions detectable amounts of substances do diffuse inward through unetched dentine in vivo. Pashley et al. (198la) demonstrated that the flux of ‘)‘I through dog dentine was similar in vivo and in vitro. It may be important that they did not remove the smear layer from the exposed dentine surface in these experiments. As a result there is likely to have been a much slower rate of outward fluid flow in vivo, and therefore less resistance to inward diffusion than would have been the case through etched dentine. Edwall and Kindlova (1971) also showed that 125Idiffused through dentine in cats, and they estimated pulpal blood flow from the rate of diffusion. Like Pashley et al. (1981a) they did not remove the smear layer from the dentine surface. Jennings and Ranly (1972) showed that less ‘*P diffused into teeth when it was applied to exposed, etched dentine in vivo in dogs than when it was applied under similar conditions to unetched dentine wit’h a smear layer. Both Fish (1933) and Bodecker and Lefkowitz (1937) found that dyes diffused into dentinal tubules in vivo, and it can be assumed that a smear layer was present in their preparations. Also, they placed the dyes under fillings, which would have further reduced the outward flow of fluid through the tubules. A flow of fluid through dentine has been demonstrated when a pressure difference was developed across the ends of the tubules in extracted human teeth (Horiuchi and Matthews, 1973; Johnson, Olgart and BrBnnstram, 19’73). In the experiments of Johnson et al. (1973) an outward flow of fluid was
645
recorded when the pressure of the pulp cavity was raised to 30mmHg, which they took to be the pressure of the pulpal tissue fluid. Pashley, Nelson and Pashley (1981b) made observations on dog teeth in vivo and recorded fluid flow through dentine when pressures of up to 240 cmH,O were applied. In experiments in progress (Vongsavan and Matthews, 1990) we have been able to record an outward flow of fluid from exposed dentine in cat teeth in vivo under physiological conditions similar to those in the present experiments where we demonstrated that Evans’ blue did not enter the tubules. The main contribution of the present experiments is to provide evidence for the first time that the velocity with which fluid flows outward through exposed dentine in vivo can be sufficient to very substantially reduce the inward diffusion of substances into the tubules. We have tested only substances with a limited range of diffusion coefficients (mol. wt 364-960) but the evidence from other work (see above) suggests that the same may apply to substances with much larger diffusion coefficients. This outward flow of fluid through exposed dentine will provide some protection against the inward diffusion of bacterial and other toxins from the mouth, although it is not enough to prevent the proliferation of bacteria through the tubules (Olga& Brannstriim and Johnson, 1974). Our findings provide an explanation for the fact that potentially damaging chemicals, such as strong acids and glutaraldehyde, can be applied to exposed dentine to facilitate the bonding of filling materials, with apparently little risk to the pulp. Brannstriim (e.g. 1987) has often emphasized that fluid flow through dentine could influence in this way the penetration of toxins and other substances. It is clear that great care has to be exercised in using extracted teeth in experiments intended to reproduce the effects of chemicals on dentine in vivo. Such experiments include studies on the mechanisms and control of caries in dentine. The penetration of acids and of proteolytic enzymes into dentinal tubules in vitro, and therefore the extent of any carious lesion, is likely to be very much greater than in vivo. Also, experiments on the bonding of filling materials to dentine may give misleading results when such tests are carried out on dried, etched dentine in vitro, as opposed to dentine in which the ends of the tubules are filled with fluid, as will probably be the state in vivo despite attempts to dry the exposed dentine surface (Tao and Pashley, 1989). When a substance is applied to exposed dentine under conditions in which there is an outward flow of fluid through the tubules, two groups of factors will determine the amount of the substance that penetrates the tubules and its concentration at different times within the tubules: those which affect the diffusion process and those which affect the mean velocity of flow. The magnitude of most of these factors is unknown, as is the precise nature of the diffusion path through the tubules. It is therefore difficult to predict the outcome under different conditions. Two factors are of particular interest in the present context: the diameter of the tubules and the tissue-fluid pressure of the pulp. In the simplest model of the system, consisting of an array of parallel-sided,
N. VONGSAVAN an d B. Mamws
646
water-filled tubules, a decrease in tubule diameter will tend to increase the concentration of the substance present after a particular time and at a particular distance along a tubule. This is because the mean velocity of flow opposing diffusion will increase in proportion to the square of the tubule diameter, whereas the concentration due to diffusion alone will be independent of tubule diameter. Thus the presence of a smear layer, or of a substance introduced in an attempt to plug the ends of the tubules, may under certain conditions increase the concentration of a substance in the tubules compared with that in open tubules. The pulpal tissue-fluid pressure will depend in part on the state of the pulpal vasculature, and therefore diffusion of substances into dentine may be affected by, for example, activity in vasomotor nerves, locally released vasoactive peptides or vasoconstrictor agents administered with local anaesthetits. The tissue-fluid pressure in normal pulp has been estimated by several techniques and a wide range of values obtained (see Heyeraas, 1985). Any inward movement of fluid through the dentinal tubules of the root after removal of the pulp (as may be predicted from the results we obtained after pulp section) could interfere with the sealing of root-canal fillings. It may therefore be advantageous to leave the smear layer that is formed on the inner dentine surface during instrumentation of the root canal. It may be possible to enable drugs (e.g. local anaesthetics, steroids and antibiotics) to penetrate dentine and gain access to the pulp by applying them to exposed dentine at a pressure of around 20 cmH*O, as was used in the present experiments to enable Evans’ blue to enter the tubules. Acknowledgement-We are very grateful to Dr W. Raab of the University of Erlangen for pointing out that cutting the root pulp may not have reduced the pressure in the corona1 pulp to atmospheric.
REFERENCES Anderson D. J. and Ronning G. A. (1962) Dye diffusion in human dentine. Archs oral Biol. 7, 505-512. Bodecker C. F. and Lefkowitz W. (1937) Concerning the “vitality” of the calcified dental tissues. J. denr. Res. 16, 463-478. Brgnnstriim M. (1987) Infection beneath composite resin restorations: can it be avoided? Oper. Dent. 12, 158-163. Edwall L. and Kindlova M. (1971) The effect of sympathetic nerve stimulation on the rate of disappearance of tracers from various oral tissues. Acta odont. stand. 29, 3874. Fish E. W. (1933) An Experimental Investigation of Enamel, Dentine and Dental Pulp. Bale, Sons and Danielsson, London.
Heyeraas K. J. (1985) Pulpal, microvascular, and tissue pressure. J. dent. Res. 64, 585-589. Holland G. R. (1975) The dentinal tubule and odontoblast process in the cat. J. Anat. 120, 169-177. Holland G. R. (1985) The odontoblast orocess: form and function. J. dent. Res. 64, 499-514. _ Horiuchi H. and Matthews B. (19731 In-vitro observations on fluid flow through human dektine caused by painproducing stimuli. Archs oral Biol. 18, 275-294. Horiuchi H. and Matthews B. (1974) Evidence on the origin of impulses recorded from dentine in the cat. J. Physiol., Land. 243, 797629. Horiuchi H. and Matthews B. (1976) Responses of intradental nerves to chemical and osmotic stimulation of dentine in the cat. Pain 2, 49-59. Iwaku M., Nakamichi I., Nakamura K., Horie K., Suizu S. and Fusayama T. (1981) Tags penetrating dentin of a new adhesive resin. Bull. Tokyo med. dent. Univ. 28, 45-51. Jennings R. E. and Ranly D. M. (1972) Autoradiographic studies of ‘*P penetration into enamel and dentin during acid etching. J. Dent. Child. 36, 69-71. Johnson G., Olgart L. and BrsnnstrBm M. (1973) Outward fluid flow in dentin under a physiologic pressure gradient: experiments in vitro. Oral Surg. 35, 238-248. Matthews B. (1976) The mechanisms of pain from dentine and pulp. Br. dent. J. 140, 574. Matthews B. and Hughes S. H. S. (1988) The ultrastructure and receptor transduction mechanisms of dentine. In Progress in Brain Research, (Eds Hamman W. and Iggo A.), Vol. 73, pp. 69-76. Elsevier, Amsterdam. Niirhi M. V. 0. and Hirvonen T. (1987) The response of dog intradental nerves to hypotonic solutions of CaCl, and other stimuli, applied to exposed dentine. Archs oral Biol. 32, 781-786. Olgart L., Brlnnstrijm M. and Johnson G. (1974) Invasion of bacteria into dentinal tubules. Acta odont. stand. 32, 61-70. Pashley D. H., Kehl T., Pashley E. and Palmer P. (1981a) Comparison of in vitro and in uiuo dog dentin permeability. J. denr. Res. 60, 763-768. Pashley D. H., Nelson R. and Pashley E. L. (1981b) In-Go fluid movement across dentine in the dog. Archs oral Biol. 26, 707-710. Pashley D. H., Tao L., Boyd L., King G. E. and Homer J. A. (1988) Scanning electron microscopy of the substructure of smear layers in human dentine. Archs oral Biol. 33, 265-270. Tao L. and Pashley D. H. (1989) Dentin perfusion effects on the shear bond strengths of bonding agents to dentin. Dent. Mater. 5, 181-184. Thomas H. F. (1979) The extent of the odontoblast process in human dentin. J. dent. Res. 58, 2207-2218. Thomas H. F. and Payne R. C. (1983) The ultrastructure of dentinal tubules from erupted human premolar teeth. J. dent. Res. 62, 532-536. Tsatas B. G. and Frank R. M. (1972) Ultrastructure of dentinal tubules near the dentino-enamel junction. Calc. Tiss. Res. 9, 238-242. Vongsavan N. and Matthews B. (1990) Fluid flow through exposed dentine in anaesthetized cats. J. Physiol., Land. 430, 37P.
0003-9969/9153.00+ 0.00 Copyright 0 1991Pergamon Press plc
THE PERMEABILITY OF CAT DENTINE AND IN VITRO
IN VW0
N. VONGSAVANand B. MATTHEWS Department of Physiology, University of Bristol, University Walk, Bristol BS8 ITD, U.K. (Accepted 19 March 1991) Summary-The apparent impermeability of dentinal tubules to chemicals applied to exposed dentine in vivo was investigated. It was shown that whereas Evans’ blue diffused readily into dentine in extracted teeth, it did not do so in visible amounts when it was applied in vim. In the invitroexperiments, decreasing the pressure of the Evans’ blue solution to 20 cmH,O below atmospheric apparently prevented the dye entering the tubules, while increasing the pressure of the solution to 15 cmH,O or more above atmospheric in vivoresulted in the dye penetrating the dentine in high concentrations. It is concluded that, in cat dentine in vim, there is an outward flow of fluid through exposed dentinal tubules, and that this flow is sufhcient to substantially reduce diffusion into the tubules of chemicals such as Evans’ blue. Key words: dentine permeability
INTIIODUCTION Anderson
and
Ronning
(1962) demonstrated
that
human dentine is freely permeable to the dye Evans’ blue. They made their observations on recently extracted teeth, and on dentine that had been exposed by fracturing off the tips of cusps. Several observations in this laboratory have suggested that cat dentine in vivo may not be as permeable as indicated by these experiments. F:or example, when solutions of cobalt chloride were applied to exposed dentine in vivo, no evidence could be found that cobalt ions diffused into the dentinal tubules or pulp (Matthews, 1976). The dentine was exposed by fracturing off approx. 1 mm from th.e tip of the cusp of a canine tooth under Ringer’s solution. This procedure was used to avoid the tubules becoming obstructed by air being sucked into them, by debris from a drill, or by a smear layer (Pashley et al., 1988). A similar result was obtained in preliminary experiments with solutions of Evans’ blue and horseradish peroxidase, and the result with the peroxidase has recently been confumed at the ultrastructural level (Matthews B. and Hughes S. H. S., unpublished observations). Further evidence that there is greater resistance to inward diffusion of substances through dentine in vivo than would be. expected from the studies on extracted teeth has been obtained in experiments on cats in which it was found that topical applications of local anaesthetic solutions or fixatives did not abolish neural responses evok’ed by mechanical stimulation of the dentine. The dentine was exposed as described above, but using a diamond disc rather than fracturing, and it was etched with acid to remove the smear layer. The only evidence that substances can diffuse through dentine in vivo has been obtained using radio-isotopes of iodine in cats (Edwall and Kindlova, 1971) and dogs (Pashley et al., 1981a) and in these instances the smear layer was not removed.
A smear layer would also have been present in the experiments of Horiuchi and Matthews (1974) in which they found that topical applications of 2% lignocaine to exposed dentine in cat teeth had no effect on the propagation of impulses in pulpal nerves. We have now investigated further the permeability of cat dentine in vitro and in vivo. MATERIALS AND
METHODS
All the observations were made on the permanent canine teeth of young adult cats. The cats were anaesthetized with sodium pentobarbitone (Sagatal; May and Baker, Dagenham, U.K. 42mg/kg intraperitoneally followed by 3 mg/kg intravenously as required). The permeability of the coronal dentine of these teeth to the dye Evans’ blue was investigated under three conditions: in teeth which had been extracted, in teeth which remained in situ with an intact blood supply, and in teeth which remained in situ but with their root pulps sectioned.
The teeth that were ex-
tracted before testing were removed surgically with minimal force. They were stored in Ringer’s solution and the dentine was exposed immediately before its permeability was investigated. Care was taken to prevent the dentine drying. In those teeth in which the pulp was sectioned, this was done in the middle third of the root after the pulp had been exposed by removing the overlying bone and dentine. There is no evidence that blood vessels enter the pulp of the cat canine other than in the apical region of the root and therefore this procedure will have stopped all blood flow to the coronal pulp. With both groups of teeth that were tested in situ, a section of dentine was removed for histological examination immediately after the period of dye application and without extracting the teeth.
641
N.
642
VONGSAVANand
B.
MATTHEWS
Further details of the methods used in individual experiments are given at the appropriate points in the Results section.
Collar
Evans’
Blue
RESULTS
Dentine exposed by fracturing
Tooth
Per-ipex
Cop
4 mm
I
Fig. 1. Scale diagram of the cap used to apply Evans’ blue to exposed dentine of cat canine teeth in vitro and in vivo.
Dentine exposed by fracturing Apart from the method of exposing the dentine, the basic preparation of the tooth was the same for all the experiments (Text Fig. 1). The surface of the enamel was cleaned and etched with 4.6 mol/l orthophosphoric acid for 30 s. Then a Perspex collar was fitted around the crown, 2-3 mm below the tip of the cusp, and sealed in place with resin (Concisea Enamel Bond System, 3M Dental Products, St Paul, MN, U.S.A.). The enamel was scored approx. 1 mm from the cusp tip and this part of the crown was fractured off with shears under Ringer’s solution. Approximately 1 mm of dentine remained over the pulp cornu. A small Perspex cap was sealed on to the collar to enclose the tip of the tooth, and filled with a solution of Evans’ blue (52 mmol/l) in isotonic saline. After 30 min, the Evans’ blue was washed out of the cap, the cap was removed, and an axial longitudinal section, approx. 0.5mm thick, was cut through the crown with a diamond disc under Ringers. The section was dehydrated overnight in 100% ethyl alcohol, cleared in methyl salicylate and examined under a microscope (Wild, type M4~) with variable dark-ground illumination. Evans’ blue is very much less soluble in ethyl alcohol or methyl salicylate than in water and therefore its diffusion through the dentine was effectively arrested when the section was placed in the alcohol. Dentine exposed with a diamond disc In some cases, both in vivo and in vitro, the same amount of tooth substance was removed from the tip of the tooth but instead of by fracturing it was removed with a diamond disc (Komet, Gebr. Brasseler, Lemgo, Germany. Type 943, thickness 0.15 mm) under a stream of Ringer’s solution. The exposed dentine surface was etched with either 4.6 mol/l ortho-phosphoric acid for 30 s or 0.1 moljl EDTA @H 7.0) for 5 min.
When Evans’ blue was applied for 30min to exposed dentine in extracted teeth (n = IO), it diffused through the opened tubules into the pulp and stained these tissues heavily [Plate Fig. 2(A)]. The teeth were examined at room temperature from 20 min to more than 10 h after extraction, having been stored in Ringer’s either at room temperature (n = 9) or frozen (n = I), and the result was the same in all cases. When the same procedures were carried out in vivo, no Evans’ blue could be detected in the dentinal tubules or pulp of the cat teeth (n = 9) [Plate Fig. 2(B)]. The most likely explanation seemed to be that there was a constant outward flow of fluid through the dentinal tubules in vim, caused by the pulpal tissuefluid pressure being above atmospheric; and that the rate of flow of this fluid was sufl’icient to severely reduce the rate of inward diffusion of the dye. We attempted to test this hypothesis by determining the effect on dye diffusion of cutting through the dental pulp near the root apex, thus arresting the flow of blood through the pulpal circulation of a tooth that was left in situ. In four teeth tested 1.5 h after cutting the root pulp, no dye could be detected in the dentine or pulp of three, but the dye reached the pulp in the other. The one tooth tested 10 h after pulp section was impermeable to the dye. We therefore concluded that fluid flowing through the dentinal tubules was probably not responsible for the lack of detectable dye penetration into the dentine. Other work in our laboratory (Matthews and Hughes, 1988) has demonstrated that the outer ends of the dentinal tubules over the pulp cornua in cat canines are not empty but contain a material that is more electron-dense than extracellular fluid when observed in stained sections in the transmission electron microscope. Similar observations have been made by others on specimens of both cat (Holland, 1975, 1985) and human (Tsatas and Frank, 1972; Thomas, 1979; Thomas and Payne, 1983) dentine. We next considered the possibility that this material might be acting as a plug, forming a barrier to diffusion; although it was not clear how it could become leaky soon after extraction of a tooth, and not do so in a tooth left in situ with its pulp cut.
Plate 1 Fig. 2. Photomicrographs of ground longitudinal sections of the crowns of cat canine teeth. The sections were dehydrated and cleared in methyl salicylate. In each case, a solution of Evans’ blue was applied for 30 min to the exposed dentine at the tip of the cusp immediately before the section was cut. The dye was applied under different conditions in each case. Original magnification: x 35. (A) Recently extracted tooth. (B) Dye applied in uivo, before the tooth was extracted. (C) Recently extracted tooth. The pressure in the cap containing the dye was maintained at ZOcmH,O below atmospheric throughout the period of dye application. (D) Dye applied in t&o. The pressure in the cap was maintained at 20cmHi0 above atmospheric.
Permeability of dentine
Plate 1
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N. VONGSAVAN and B. MATTHEWS
Autolysis would be expected to proceed similarly under both conditions. Maybe the act of extraction, despite our care to minimize the trauma to the tooth, was causing the plugs to move in the tubules and to become leaky, or perhaps the temperature of the tooth during the period between the arrest of the circulation and testing was an important factor. The latter possibility was tested first. Extracted teeth were stored at either 4 (n = 6) or 37°C (n = 4) for periods from 20 min to 10 h before the permeability of the dentine was tested in vitro. All these teeth were found to be permeable to Evans’ blue. Some teeth were also left in situ with their pulps intact and tested after death of the animal. The body was stored at room temperature until the teeth were tested. The dye penetrated the dentine 1.5 h after death (n = 2), but none was detectable 12 h after (n = 3). Thus while the results obtained from teeth both in vivo and in vitro after extraction were very reproducible, those obtained from teeth in situ after arrest of pulpal blood flow were very variable and did not fit into any obvious pattern. The only ways in which we could imagine that the surgical extraction of a tooth might dislodge plugs in the dentinal tubules over the pulp cornua were either if negative pressures were developed in the pulp during the extraction or if tension was applied to the odontoblast processes via connective tissue, nerves and vessels that passed through the apical foramen. Both mechanisms seemed improbable but were tested by opening the pulp cavity in the root and cutting through the pulp before the tooth was extracted. The dentine was permeable to Evans’ blue in all cases (n = 3), despite these precautions. We next considered the possibility that changes in the surface charge on the lining of the dentinal tubules was affecting their permeability to the dye. Evans’ blue is an acid dye (mol. wt 960) and therefore a change from a negative to a positive surface charge would tend to impede its penetration. This was tested by using the basic dyes Janus green (mol. wt 483) and methylene green (mol. wt 364). Methylene green has the advantage that, like Evans’ blue, it is relatively insoluble in alcohol. Neither Janus green (n = 1) nor methylene green (n = 4) penetrated dentine in vivo, but both dyes reached the pulp in vitro (n = 1 in both cases). Thus the surface charge on the inner surface of the tubules was not responsible for the differences observed. We finally returned to our original, fluid-flow hypothesis. Our test of this hypothesis would not have been valid if, for some reason, a significant pressure tending to force fluid out through the dentine remained in the coronal pulp after cutting the root pulp, but not after the extraction of a tooth. One possible mechanism which was considered was that at the time of pulp section in the root (but not for some reason after extraction), blood vessels there were occluded by vasoconstruction and clot formation, and that as a result pressure was maintained for some time in the vessels in the coronal pulp on account of its low compliance (Heyeraas K. J., 1990, personal communication). This possibility seems to have been eliminated, however, by the observation that dye
entered the dentine of extracted teeth in which the root pulp had been sectioned just before extraction (see above). An alternative possibility was that the pressure in the coronal pulp of a tooth in situ was maintained after section of the root pulp as a result of communication between the tissue fluid in the pulp and that in the periodontal tissues (in which the blood flow was largely intact) through the dentinal tubules and cementum of the root. The fluid-flow hypothesis was therefore tested in an alternative way, by increasing the hydrostatic pressure of the Evans’ blue in vivo and by decreasing it below atmospheric in vitro. When the pressure in the cap was reduced to 15 cmH,O below atmospheric in vitro, visible amounts of the dye still entered dentine (n = 2); but no dye was detectable after exposure at a pressure of -20 cmH,O (n = 6) [Plate Fig. 2(C)]. When the cap pressure was increased to lOcmH,O above atmospheric in vivo, Evans’ blue was not detectable in the dentine (n = 2), but it was visible with pressures of 15 and 20 cmH,O (n = 7 and 2, respectively) [Plate Fig. 2(D)]. Dentine exposed with a diamond disc
When dentine was tested after it had been exposed in vitro by cutting with a diamond disc instead of by
fracturing, no Evans’ blue could be detected in the tubules (n = 2). However, when the exposed dentine was etched with orthophosphoric acid or EDTA before testing, the dye entered the tubules and stained the pulp in all six teeth examined. These teeth were examined at room temperature and between 20min and 24 h after extraction. Dentine tested in vivo after it had been exposed with a diamond disc and etched with orthophosphotic acid, contained no detectable Evans’ blue (n = 3). The same result was obtained in one tooth that was etched with EDTA. DlSCUSSlON
Our observations on the permeability of dentine to Evans’ blue in recently extracted cat teeth agree with previous findings on human teeth (Anderson and Ronning, 1962). Under these conditions, as long as the tubules were not obstructed by a smear layer, Evans’ blue diffused rapidly through the dentine. When Evans’ blue was similarly applied to exposed dentine in vivo, none appeared to enter the dentine. This same result was obtained whether the dentine had been exposed by fracturing or by using a diamond disc, provided that the smear layer left by the disc was first removed by etching. The results obtained by changing the pressure gradient across the dentine, both in vitro and in vivo, indicated that the rate of outward flow of fluid through dentinal tubules determined the rate at which dye was able to diffuse upstream into them. Thus, we conclude that when dentine is exposed in vivo, there is an outward flow of fluid through the tubules and that this flow is sufficiently rapid to severely limit the rate of inward diffusion of chemicals. After extraction of a tooth, the pulpal tissue-fluid pressure falls and under these
Permeability of dentine conditions the rate of outward flow of fluid through the tubules decreases to the point that it no longer affects the rate of inward diffusion of the dye. In all of these. studies, the Evans blue solution would have added an osmotic effect, which would have tended to increase the outward flow of fluid. This is unlikely to have influenced the results significantly, however, in view of the low molar concentration of the dye and the fact that even in vivo it was shown that the dye was not prevented from entering the tubules by virtue of the size of its molecule. All of our findings can be explained on the basis of such a mechanism except those obtained from teeth left in situ but with no pulpal blood flow. The only explanation we can offer for the differences between these results and those obtained from extracted teeth is that, after section of the pulp of a tooth left in situ, the tissue-fluid pressure in the coronal pulp tended to be maintained at a hi,gher level than in extracted teeth as a result of communication with the tissue fluid of the periodontal ligam.ent through the dentinal tubules of the upper part of the tooth. Our findings also explain earlier ones which suggested that cat dentine in vivo was impermeable to substances such as horseradish peroxidase and local anaesthetics (see Introduction). They also account for other observations, including the following. It has been shown that nerve endings in teeth are not excited when chemical stimuli are applied to dentine unless only a thin layer of dentine remains over the pulp (Horiuchi and Matthews, 1976; NIhri and Hirvonen, 1987). Iwaku et al. (1981) reported that resin penetrated the ends of tubules more deeply when it was applied to etched dentine in vitro than in vivo. There is evidence that under certain conditions detectable amounts of substances do diffuse inward through unetched dentine in vivo. Pashley et al. (198la) demonstrated that the flux of ‘)‘I through dog dentine was similar in vivo and in vitro. It may be important that they did not remove the smear layer from the exposed dentine surface in these experiments. As a result there is likely to have been a much slower rate of outward fluid flow in vivo, and therefore less resistance to inward diffusion than would have been the case through etched dentine. Edwall and Kindlova (1971) also showed that 125Idiffused through dentine in cats, and they estimated pulpal blood flow from the rate of diffusion. Like Pashley et al. (1981a) they did not remove the smear layer from the dentine surface. Jennings and Ranly (1972) showed that less ‘*P diffused into teeth when it was applied to exposed, etched dentine in vivo in dogs than when it was applied under similar conditions to unetched dentine wit’h a smear layer. Both Fish (1933) and Bodecker and Lefkowitz (1937) found that dyes diffused into dentinal tubules in vivo, and it can be assumed that a smear layer was present in their preparations. Also, they placed the dyes under fillings, which would have further reduced the outward flow of fluid through the tubules. A flow of fluid through dentine has been demonstrated when a pressure difference was developed across the ends of the tubules in extracted human teeth (Horiuchi and Matthews, 1973; Johnson, Olgart and BrBnnstram, 19’73). In the experiments of Johnson et al. (1973) an outward flow of fluid was
645
recorded when the pressure of the pulp cavity was raised to 30mmHg, which they took to be the pressure of the pulpal tissue fluid. Pashley, Nelson and Pashley (1981b) made observations on dog teeth in vivo and recorded fluid flow through dentine when pressures of up to 240 cmH,O were applied. In experiments in progress (Vongsavan and Matthews, 1990) we have been able to record an outward flow of fluid from exposed dentine in cat teeth in vivo under physiological conditions similar to those in the present experiments where we demonstrated that Evans’ blue did not enter the tubules. The main contribution of the present experiments is to provide evidence for the first time that the velocity with which fluid flows outward through exposed dentine in vivo can be sufficient to very substantially reduce the inward diffusion of substances into the tubules. We have tested only substances with a limited range of diffusion coefficients (mol. wt 364-960) but the evidence from other work (see above) suggests that the same may apply to substances with much larger diffusion coefficients. This outward flow of fluid through exposed dentine will provide some protection against the inward diffusion of bacterial and other toxins from the mouth, although it is not enough to prevent the proliferation of bacteria through the tubules (Olga& Brannstriim and Johnson, 1974). Our findings provide an explanation for the fact that potentially damaging chemicals, such as strong acids and glutaraldehyde, can be applied to exposed dentine to facilitate the bonding of filling materials, with apparently little risk to the pulp. Brannstriim (e.g. 1987) has often emphasized that fluid flow through dentine could influence in this way the penetration of toxins and other substances. It is clear that great care has to be exercised in using extracted teeth in experiments intended to reproduce the effects of chemicals on dentine in vivo. Such experiments include studies on the mechanisms and control of caries in dentine. The penetration of acids and of proteolytic enzymes into dentinal tubules in vitro, and therefore the extent of any carious lesion, is likely to be very much greater than in vivo. Also, experiments on the bonding of filling materials to dentine may give misleading results when such tests are carried out on dried, etched dentine in vitro, as opposed to dentine in which the ends of the tubules are filled with fluid, as will probably be the state in vivo despite attempts to dry the exposed dentine surface (Tao and Pashley, 1989). When a substance is applied to exposed dentine under conditions in which there is an outward flow of fluid through the tubules, two groups of factors will determine the amount of the substance that penetrates the tubules and its concentration at different times within the tubules: those which affect the diffusion process and those which affect the mean velocity of flow. The magnitude of most of these factors is unknown, as is the precise nature of the diffusion path through the tubules. It is therefore difficult to predict the outcome under different conditions. Two factors are of particular interest in the present context: the diameter of the tubules and the tissue-fluid pressure of the pulp. In the simplest model of the system, consisting of an array of parallel-sided,
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water-filled tubules, a decrease in tubule diameter will tend to increase the concentration of the substance present after a particular time and at a particular distance along a tubule. This is because the mean velocity of flow opposing diffusion will increase in proportion to the square of the tubule diameter, whereas the concentration due to diffusion alone will be independent of tubule diameter. Thus the presence of a smear layer, or of a substance introduced in an attempt to plug the ends of the tubules, may under certain conditions increase the concentration of a substance in the tubules compared with that in open tubules. The pulpal tissue-fluid pressure will depend in part on the state of the pulpal vasculature, and therefore diffusion of substances into dentine may be affected by, for example, activity in vasomotor nerves, locally released vasoactive peptides or vasoconstrictor agents administered with local anaesthetits. The tissue-fluid pressure in normal pulp has been estimated by several techniques and a wide range of values obtained (see Heyeraas, 1985). Any inward movement of fluid through the dentinal tubules of the root after removal of the pulp (as may be predicted from the results we obtained after pulp section) could interfere with the sealing of root-canal fillings. It may therefore be advantageous to leave the smear layer that is formed on the inner dentine surface during instrumentation of the root canal. It may be possible to enable drugs (e.g. local anaesthetics, steroids and antibiotics) to penetrate dentine and gain access to the pulp by applying them to exposed dentine at a pressure of around 20 cmH*O, as was used in the present experiments to enable Evans’ blue to enter the tubules. Acknowledgement-We are very grateful to Dr W. Raab of the University of Erlangen for pointing out that cutting the root pulp may not have reduced the pressure in the corona1 pulp to atmospheric.
REFERENCES Anderson D. J. and Ronning G. A. (1962) Dye diffusion in human dentine. Archs oral Biol. 7, 505-512. Bodecker C. F. and Lefkowitz W. (1937) Concerning the “vitality” of the calcified dental tissues. J. denr. Res. 16, 463-478. Brgnnstriim M. (1987) Infection beneath composite resin restorations: can it be avoided? Oper. Dent. 12, 158-163. Edwall L. and Kindlova M. (1971) The effect of sympathetic nerve stimulation on the rate of disappearance of tracers from various oral tissues. Acta odont. stand. 29, 3874. Fish E. W. (1933) An Experimental Investigation of Enamel, Dentine and Dental Pulp. Bale, Sons and Danielsson, London.
Heyeraas K. J. (1985) Pulpal, microvascular, and tissue pressure. J. dent. Res. 64, 585-589. Holland G. R. (1975) The dentinal tubule and odontoblast process in the cat. J. Anat. 120, 169-177. Holland G. R. (1985) The odontoblast orocess: form and function. J. dent. Res. 64, 499-514. _ Horiuchi H. and Matthews B. (19731 In-vitro observations on fluid flow through human dektine caused by painproducing stimuli. Archs oral Biol. 18, 275-294. Horiuchi H. and Matthews B. (1974) Evidence on the origin of impulses recorded from dentine in the cat. J. Physiol., Land. 243, 797629. Horiuchi H. and Matthews B. (1976) Responses of intradental nerves to chemical and osmotic stimulation of dentine in the cat. Pain 2, 49-59. Iwaku M., Nakamichi I., Nakamura K., Horie K., Suizu S. and Fusayama T. (1981) Tags penetrating dentin of a new adhesive resin. Bull. Tokyo med. dent. Univ. 28, 45-51. Jennings R. E. and Ranly D. M. (1972) Autoradiographic studies of ‘*P penetration into enamel and dentin during acid etching. J. Dent. Child. 36, 69-71. Johnson G., Olgart L. and BrsnnstrBm M. (1973) Outward fluid flow in dentin under a physiologic pressure gradient: experiments in vitro. Oral Surg. 35, 238-248. Matthews B. (1976) The mechanisms of pain from dentine and pulp. Br. dent. J. 140, 574. Matthews B. and Hughes S. H. S. (1988) The ultrastructure and receptor transduction mechanisms of dentine. In Progress in Brain Research, (Eds Hamman W. and Iggo A.), Vol. 73, pp. 69-76. Elsevier, Amsterdam. Niirhi M. V. 0. and Hirvonen T. (1987) The response of dog intradental nerves to hypotonic solutions of CaCl, and other stimuli, applied to exposed dentine. Archs oral Biol. 32, 781-786. Olgart L., Brlnnstrijm M. and Johnson G. (1974) Invasion of bacteria into dentinal tubules. Acta odont. stand. 32, 61-70. Pashley D. H., Kehl T., Pashley E. and Palmer P. (1981a) Comparison of in vitro and in uiuo dog dentin permeability. J. denr. Res. 60, 763-768. Pashley D. H., Nelson R. and Pashley E. L. (1981b) In-Go fluid movement across dentine in the dog. Archs oral Biol. 26, 707-710. Pashley D. H., Tao L., Boyd L., King G. E. and Homer J. A. (1988) Scanning electron microscopy of the substructure of smear layers in human dentine. Archs oral Biol. 33, 265-270. Tao L. and Pashley D. H. (1989) Dentin perfusion effects on the shear bond strengths of bonding agents to dentin. Dent. Mater. 5, 181-184. Thomas H. F. (1979) The extent of the odontoblast process in human dentin. J. dent. Res. 58, 2207-2218. Thomas H. F. and Payne R. C. (1983) The ultrastructure of dentinal tubules from erupted human premolar teeth. J. dent. Res. 62, 532-536. Tsatas B. G. and Frank R. M. (1972) Ultrastructure of dentinal tubules near the dentino-enamel junction. Calc. Tiss. Res. 9, 238-242. Vongsavan N. and Matthews B. (1990) Fluid flow through exposed dentine in anaesthetized cats. J. Physiol., Land. 430, 37P.
