fluid flow through human dentine

.4rcfrs oral Bml. Vol.12, pp.209-216, 1967. Pergamon Pm5 Ltd. Printed in Gt. Britain.

FLUID FLOW THROUGH HUMAN DENTINE

D. J. ANDERSON,* B. MATTHEwst and C. GORRETTA:

Department of Physiology, Guy’s Hospital Medical School, London, S.E.1

Summary-With freshly extracted sound teeth it has been shown that a saturated solution of CaCI, at 37°C causes fluid movement through dentine when applied to a small occlusal cavity. Measurement of the rate of flow was made photographically and the maximum value obtained in a group of eight teeth was 4.8 n&c. During a sequence of eight applications, the rate of flow declined. When two cavities were cut in the same tooth and the CaCI, was applied 8 times in succession to one of the cavities, and the sequence was repeated on the second cavity, a similar trend of declining flow was seen from both cavities.

ANDERSON and RONNING (1962) and ANDERSON, MATTHEWS and SHELTON (1966) showed that the number reports of pain during repeated osmotic stimulations declined in single and in two cavity experiments. Taken together the results from in vitro and in viva experiments suggest that with osmotic stimulation (1) flow through dentine and pain production are related events and (2) the decline in the number of reports of pain during repeated stimulation is due to some change in the dentine, rather than in the pulp.

INTRODUCTION

THE ABILITY of various solutions to cause pain when applied to cavities cut in human

dentine appears to be related to their osmotic pressure (ANDERSON and RONNING,

1962; MATTHEWS, 1966). The means whereby pain is evoked from dentine by osmotic

and other stimuli is unknown and with the object of shedding light on this problem, an

attempt has been made to investigate the physical changes produced in dentine by

osmotic stimulation. When a solution exerting a high osmotic pressure is applied to a

cavity in dentine it seems likely that the high concentration of solute would cause

water to move towards the cavity from the dentinal tubules and pulp. Experiments

have been performed to detect and measure the movements of water through dentine

in vitro in response to solutions known to cause pain and this report deals with the

first of these experiments.

MATERIALS AND METHODS

The method has already been described (ANDERSON, MAITHEWS and GORRETTA,

1966). The teeth used were sound premolars extracted for orthodontic reasons.

Immediately after extraction they were stored in physiological saline, and within a

few hours a small cavity or two small cavities were cut through the occlusal surface,

just into the dentine. Cavity preparation was carried out with a slow-running tungsten

Present addresses: *The University of Bristol Dental School, Lower Maudlin Street, Bristol 1. TDepartment of Physiology, The Medical School, University Walk, Bristol 8. iuniversity of Oregon Dental School, Sam Jackson Park, Portland 1, Oregon, U.S.A.

209

210 D. .I. ANDERSON, B. MATTHEWS AND C. GORRETTA

carbide bur under a stream of cold saline. After cavity preparation, the cavity was kept moist with a pledget of cotton wool soaked in saline, while the other preparatory manipulations were made. The root of the tooth was cut off just beyond the limit of the enamel cap and the coronal pulp tissue removed with a probe. This usually brought away the tissue in a single piece and although histological examination was not performed in these experiments, it has been observed by us previously and by others (TEN CATE and SHELTON, 1966) that removal of pulp tissue in this way frequently leaves the odontoblast layer almost intact adhering to the dentine. After removal of the pulp tissue, the pulp cavity was filled with saline.

The next step was to fit the tooth crown into a Perspex holder which was a short piece of rod in which a depression had been cut to receive the crown. Into the bottom of the depression had been sealed a narrow glass capillary of internal diameter 0.3 mm and 3 cm in length. The operation of inserting the tooth into the holder took place in a bath of saline and thus fluid continuity was established from the dentinal tubules, through the pulp cavity, to the capillary tube. The junction between the tooth and holder was sealed with wax (see Fig. 1). With this arrangement, it was

--Perspex holder

--Copillory

-Fluid meniscus

FIG. 1. Diagram of tooth crown in Perspex holder.

expected that fluid movement through dentine and pulp would be revealed by movement of the meniscus in the capillary tube. The apparatus is shown diagrammatically in Fig. 2. The tooth and attached capillary were mounted on a frame fixed to a microscope mechanical stage.

The method chosen to detect movement of the capillary meniscus was to illuminate it obliquely, magnify the spot of light so produced and record its movement on moving photographic film. Light reflected from the meniscus passed through a lens system on to a hinged mirror from which it was further reflected on to a ground glass screen for

FLUID FLOW THROUGH HUMAN DENTINE

FIG. 2. Diagram of apparatus for recording fluid movement. Apparatus shown in plan.

211

focussing. The mirror could be moved out of the light path, thereby allowing the light from the meniscus to pass through a slit and fall on moving photographic film. The position of the camera was arranged so that light focussed on the ground glass screen also came to focus on the film. The film moved at right angles to the capillary and therefore movement of the meniscus produced a sloped line on the photographic record; the steepness of the slope depending on the rate of movement of the meniscus and of the film. Two small light bulbs attached close to the film provided time and event records on the photographic tracing (see Fig. 3).

It was necessary to ensure that the environmental temperature of the tooth re- mained constant, and this was achieved by enclosing the tooth and supporting apparatus in a Perspex box. Temperature stability in the box was maintained by a modified hair drier in which the temperature of the heating element was controlled by the operator with a rheostat. The air i&t of the hair drier was adapted to take a wide bore flexible tube attached to the Perspex box and the outlet of the drier was also attached to the box; thus a continuous circulation of air took place and maintained the box temperature at 37”Ci 1°C. All solutions were heated to 37°C before the experiment began and then placed in the box, and manipulations were performed with one hand inserted into the box through a rubber sleeve.

Immediately after inserting the tooth into the apparatus, the volume of the pulp and capillary contents underwent a change due to the alteration in environmental temperature. Stabilization was slow, but was judged to have been achieved when the meniscus showed no movement for a period of several minutes. In all the experiments to be reported, saturated CaCl, at 37°C was used’as the test solution. This had been the most effective of the range of solutions previously found to cause pain and was therefore considered to be the one most likely to produce a detectable fluid movement. Again, since eight applications in succession, each lasting for 30 set, had been a common procedure in viva this was chosen for the experiments in vitro. After every application, the cavity was first rinsed with saline, and then cleared of fluid with a fine bore tube connected to a vacuum pump. For control purposes, the sequence of eight applications of saturated CaCI, was preceded by one application of physiological saline for 30 sec.

212 D. J. ANDERSON, B. MATTHEWS AND C. GORRETTA

Two experiments have been performed with this technique. The object of the first was merely to discover whether or not saturated CaCl, at 37°C caused fluid movement through dentine and to measure the rate of movement if it occurred. The second experiment was designed to investigate the pattern of fluid movement with repeated applications of CaCl,. It had been found that repeated osmotic stimulation caused a decline in the number of reports of pain (ANDERSON, MATTHEWS and SHELTON, 1966) and on the basis or results of experiments in which two cavities in the same tooth were tested with the same sequence of eight stimuli first to one cavity then to the next, it was inferred that a local change in the dentine under stimulation or in the immediately subjacent pulp was responsible for the decline in the number of reports of pain. To test this inference further, exactly the same experimental procedure was adopted in vitro, save for the use of one concentration of CaCl, eight times (saturated at 37°C) instead of four different concentrations, each one twice. Two cavities were cut in the occlusal surface of a premolar and the sequence of eight applications of saturated CaCI, was made to one of the cavities while leaving the other untouched. Then the same sequence was applied to the second cavity.

RESULTS

Experiment 1. Fluid jio~? resulting,from eight applications of a saturated solution of

CaCI, at 37°C to one cavity

Eight sound premolar teeth were used in this experiment, the results of which are shown in Fig. 4. Flow is expressed in nanolitres/second and the diagram shows mean values rt S.E. The maximum flow obtained in this series was 4.8 nl. There is a decline in rate of flow during the experiment.

1 2 3 4 5 6 7 8 Position in sequence

FIG. 4. Mean flow in response to eight applications of saturated CaCI, at 37°C to dentine in eight teeth. Vertical lines about each point represent standard error.

FLUID FLOW THROUGH HUMAN DENTINE 213

Experiment 2. Fluid flow resulting from eight applications of a saturated solution of C&l, at 37°C to two cavities

Figure 5 shows results obtained from two cavities in each of the eight teeth. The graph shows mean values * S.E. from each of the eight applications. Inspection of the results suggests that those from Cavity 2 are larger than those from Cavity 1.

Cavity 1.

3’51-----

Position in sequence

Cavity 2.

3.5r---

FIG. 5. Mean flow from two cavities in each of eight teeth. The sequence of eight applications of saturated CaCl, was made to cavity 1 before cavity 2.

Statistical analysis showed that the mean flows obtained from Cavity 2 were greater than from Cavity 1 following applications 1, 2, 3 and 5, whereas the remainder were not significantly different at the one in twenty level. The maximum flow in this series was 4.1 nl/sec.

DISCUSSION

It was not surprising that saturated CaCl, at 37°C caused fluid movement through the dentinal tubules when applied to the floor of a cavity in dentine. However, the decline in flow during a series of repeated applications of the solution was unexpected and so far we have no experimental evidence on which to build a satisfactory hypothesis to account for it. Figure 6 shows results from experiments in vivo in which solutions exerting osmotic pressures in the range 200-2800 atm have been applied repeatedly to dentine. These results, which are taken from a series to be published,


lO(

N

6(

4(

2c

c

)-

I-

l-

,_ I I I I I I

2 3 4 6

Position in sequence

FIG. 6. Reports of pain as a percentage of the applications of CaCl, solutions in the range 200-2800 atm. Results from ninety-five subjects (to be published in detail).

and which confirm earlier findings in a smaller series (ANDERSON and RONNING, 1962)

show the decline in pain-producing effect with repeated applications and follow a trend similar to that shown by flow in Fig. 4. Although the two sets of results are not strictly comparable, since those in Fig. 6 were obtained with various concentrations of CaCl, over the range of 200-2800 atm whereas the flow results were obtained only with a saturated solution at 37°C with an osmotic pressure of about 2800 atm, never- theless the similarity is undeniable.

Figure 7 shows results of experiments already published (ANDERSON et al., 1966) in which the number of reports of pain declined in two cavities in the same tooth. It was suggested that these results supported the view that the decline in the number of reports of pain was due to some change either in the dentine or in the immediately subjacent pulp. Further light can be thrown on this hypothesis by the results of the present experiments, since they follow the same pattern. The main mass of pulp tissue had been removed in preparing the tooth for these experiments and it therefore follows that the decline in flow was due to change in the dentine or odontoblasts still attached to it. The decline in sensitivity could have had the same cause and the phrase “immediately subjacent pulp” can be narrowed to mean odontoblast layer.

The main conclusion from this investigation is that fluid movement through dentine and pain production are related phenomena. The nature of the relationship is not yet clear. It might be, as suggested by BRXNNSTRGM and ASTRGM (1964) and BRXNNSTRGM (1966), that the pulp is a mechanoreceptor ; presumably it is stimulated by pressure changes. The fact that flow can be demonstrated in vitro suggest that pressure changes could occur in vivo.

FLUID FLoW THROUGH HUMAN DENTINE 215

CAVITY 1 CAVITY 2

20 -

10. 1 1 1 1 I I . 1 2 3 4 5 6 7 8

Stimulus sequence Stimulus sequence

FIG. 7. Reports of pain as a percentage of applications of CaCI, to two cavities in the same tooth. The sequence of eight stimulations was applied to Cavity 1 before Cavity 2. Results from eight subjects (from ANDERSON et al., 1966).

BR~NNSTR~~M (1966) has recorded both flow and pressure changes in Vitro resulting from procedures known to cause pain. His experimental technique was similar to that described in the present report and he regarded his results as providing further

support for his mechanoreceptor hypothesis. Our results are also compatible with this hypothesis, but other hypotheses must be considered before BRKNNSTR~~M’S can be accepted. Fluid movement, pressure changes and aspiration of odontoblasts are all indications of a disturbance transmitted to the pulp. It does not necessarily follow that the receptors stimulated by this disturbance are mechanoreceptors. Odontoblast aspiration almost certainly results in damage to the cells and would

therefore cause the liberation of injury products such as histamine and 5-hydroxy- tryptamine. These could stimulate nociceptor endings in the pulp or even in the dentinal tubules. However, if future work confirms the pulpal mechanoreceptor hypothesis, the final form it takes must be influenced by our findings, which show that the sensitivity of the receptor mechanism and the magnitude of physical disturbances transmitted to it can be affected by changes in the dentine and odontoblasts.

Acknowledgements-This investigation was supported by U.S.P.H.S. Research Grant D1037 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, U.S.A.

RCsum&--Une solution saturkc de Ca Cl* a 37” C, appliquee a une petite cavite occlusale, sur des dents fraichement extraites, provoque un mouvement liquidien a travers la dentine. La mesure de la vitesse d’ecoulement est realide photographiquement et la valeur la plus eleve obtenue dans un groupe de 8 dents est de 4,8 nl/sec. Apre une serie de huit applications, la vitesse d’ecoulement deminue. Lorsque deux cavites sont


prCparees dans la mEme dent et que le Ca Cl, est applique 8 fols de suite dans l’une des cavit&, et que l’on recommence la s&ie au niveau de la seconde cavit8, le m&me type de d&croissance est observe dans les 2 cavites.

ANDERSON~~RONNING (1962)et ANDERSON, M~~~~~~~etS~~~~0~(1966)ontmontrt+ que le nombre de douleurs ressenties au tours de stimulations osmiques r&p&es dCcroit dans des expkriences portant sur une seule et deux cavitts. De I’ensemble des rCsultats des exptriences in vitro et in vivo par stimulations osmotiques, il ressort que (1) que I’Ccoulement d travers dentine et la douleur sont des faits lits et (2) que la diminution due nombre des douleurs ressenties, au tours de stimulations rtp&es, est ii& a des changements dans la dentine plut6t due dans la pulpe.

Zusammenfassung-Mit frisch extrahierten gesunden Zghnen konnte gezeigt werden, da13 eine gesPttigte CaCl,-LGsung bei 37’C eine Fliissigheitsbewegung durch das Dentin verursacht, wenn sie in einer kleinen okklusalen Kavitlt angewandt wird. Die FlieR- geschwindigkeit wurde photographisch gemessen; das in einer Gruppe von 8 Zghnen beobachtete Maximum betrug 4,8nl/sec; wlhrend 8 aufeinanderfolgenden Appli- kation nahm die FlieBgeschwindikgiet ab. Wenn zwei Kavitlten in dense1 ben Zahn prlpariert wurden und das CaCl, 8 ma1 hintereinander in der einen Kavitat angewandt wurde, undwenn diese Folgein der zweiten Kavitlt wiederholtwurde, IieR sich beibeidenKavitMeneinlhnlicherEffektabnehmenderFliel3geschwindigkeitbeobachten.

ANDERSON und RONNING (1962) und ANDERSON, MATTHEWS und SHELTON (1966) haben gezeigt, daR die Anzal von Schmerzreaktionen nach wiederholten osmotischen Stimulationen in Experimenten writ einer und mit zwei Kavitgten abnahmen. In vitro und in vivo-Versuche deuten zusammengenommen darauf hin, da8 die Fliissigkeits- bewegung durch das Dentin und die Schmerzentwicklung mit osmotischen Stimula- tionen (1) zusammenhgngende Ereignisse sind, und daR (2) die Abnahme der Anzahl von Schmerzreaktionen wghrend wiederholter Stimulation eher auf irgendeine Veriinde- rung im Dentin als in der Pulpa zuriickzufiihren ist.

REFERENCES

ANDERSON, D. J., MATTHEWS, B. and GORRETTA, C. 1966. Fluid movement through human dentine. J. dent. Res. Proc. British Division.

ANDERSON, D. J., MATTHEWS, B. and SHELTON, L. E. 1967. Variations in the sensitivity to osmotic stimulation of human dentine. A&s oral Biol. 12,43.

ANDERSON, D. J. and RONNING, G. A. 1962. Osmotic excitants of pain in human dentine. Archs oral biol. 1, 513-523.

BR.&NNSTR&, M. and ASTR~~M, A. 1964, Study on the mechanism of pain elicited from the dentine. J. dent. Res. 43,619-625.

BR~NNSTR~M, M. 1966. The hydrodynamics of the dental tubule and of pulp fluid: its significance in relation to the sensitivity of dentine. Proc. ORCA Congr. 16-18.

MATTHEWS, B. 1966. The distribution of dental pain thresholds. J. dent. Res. Proc. British Division. TEN CATE, A. R. and SHELTON, L. E. 1966. Cholinesterase activity in human teeth. Archs oral

Biol. 11,423-428.

FLUID FLOW THROUGH HUMAN DENTIN!?

FIG. 3. Records 1, 3, 5 and 8 from a series of eight applications of saturated CaCI, to a cavity in dentine. On each record: Upper tracing: movement of capillary meniscus, the steepness of the slope is proportional to the rate of flow. Middle tracing: event marker, indicating the period of application of CaCI, Lower tracing: time in 5-set intervals.

PLATE 1

A.0.B.f.p.216

fluid flow through human dentine

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