Arch oral Bid. Vol. 35, No. 7, pp. 523-527. Printed in Great Britain. All rights reserved
EVAPORATIVE
1990 Copyright
WATER
LOSS FROM IN VITRO
HUMAN
0003-9969/90 $3.00 + 0.00 % 1990 Pergarbn Press plc
DENTINE
H. E. GOODIS,’ L. TAO’ and D. H. PASHLEY’ ‘Departmentof Restorative Dentistry, University of California, San Francisco, CA 94143 and ‘Department of Oral Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A. (Accepted 30 January 1990) Summary-The rate of spontaneous evaporation of water from dentine was measured in extracted human teeth in vitro. !;pontaneous water loss was the same with or without a smear layer. When air was blown on the dentine, the rate of evaporation increased significantly. After removal of the smear layer, the air blast-induced evaporative loss was twice as great as before its removal. Thus, with a smear layer present, evaporation is the major route by which fluid is lost from dentine rather than by filtration of dentinal fluid. After smear layer removal, fluid filtration sometimes may exceed the spontaneous rate of fluid evaporation. Key words:
evaporation,
dentinal
fluid, dentine,
filtration,
dentine
sensitivity,
MATERIALS
IhTRODUCTION
Crown segment
During many dental procedures large amounts of dentine are exposed by removal of peripheral hard tissues. This provides the potential for movement of pulpal fluid to the surface. There are two mechanisms by which fluid so moves, convection and evaporation. The rat12of convection is the product of the pulpal tissue pressure and the hydraulic conductance of the dentine (Reeder et al., 1978; Fogel, Marshall and Pashley, 1988). Dentine covered with a smear layer has a relatively low hydraulic conductance. Even in the absence of a smear layer, the rate of fluid flow through dentinal tubules brought about by filtration of pulpal fluid down existing hydrostatic pressure gradients is usually too low to stimulate pulpal mechanoreceptors via hydrodynamic effects. H:owever, when such exposed dentine is subjected to a blast of air, water evaporates from it at a rate which can cause pain (Brannstrom and Astrom, 1964; Brannstriim and Johnson, 1978). Nothing is known about the relative rates of spontaneous evaporation from dentine in the presence or absence of smear layers or how much evaporation increases following air blasts. BrHnnstriim and Astriim (1964) demonstrated that air blasts to dentine cause a measurable amount of water evaporation from dentine. Their h vitro system was not sensitive enough to measure spontaneous evaporation, although in vivo allowing dentine to evaporate spontaneously to dryness caused pain (Brannstrom and Johnson, 1978). Our purpose was to develop a method for quantifying the evaporation of water from dentine surfaces in vitro, in the presence and absence of smear layers and in response to air blasts, and to compare those fluid fluxes with fluid filtration rates.
hydrodynamic
theory.
AND METHODS
preparation
Six extracted, unerupted third molars were used. Immediately after extraction, they were placed in an isotonic saline solution (phosphate-buffered saline) containing 0.2% sodium azide to prevent bacterial growth, and stored at 4°C for less than 1 month. Crown segments were prepared in the following manner. The occlusal surface of the tooth was attached to a plastic mount with an epoxy resin cement (Epoxy, Cole Palmer, Chicago, IL, U.S.A.). The mount was placed in a holding device attached to a cutting saw (Isomet, Buehler Ltd, Lake Bluff, IL, U.S.A.). Two parallel cuts were made with a diamond blade under water coolant. The first cut removed the roots near the cementumenamel junction and the second removed the cusps and the occlusal enamel (Fig. 1). This produced a crown segment which contained the top of the coronal pulp chamber and a flat occlusal surface of dentine with no enamel except around the periphery. Any pulpal tissue remaining was removed, with care taken not to touch the dentine within the pulp chamber. Plexiglas squares (2 x 2 x 0.5 cm) were prepared with holes drilled through their centres. A 2-cm 18 ga stainless-steel tube was passed through the hole to end flush with one flat surface and to extend past the opposite surface (Fig. 1). The crown segment was then attached to the Plexiglas square with a cyanoacrylate adhesive (Zapit, DVA, Yorba Linda, CA, U.S.A.). At the end of experiments, the crowns were sectioned longitudinally to permit measurement of the thickness of dentine remaining between the highest pulp horn and the cut surface. This value ranged from 0.5 to 0.7 mm (0.5 f 0.2 f f SD, N = 6). 523
524
H. E. GOODIS er al. Crown segment 100mm
long, 34
capacity
micropipette
Microsyringe
Fig.
1. Schematic
diagram
of the tooth
preparation water
Apparatus
Two versions of an apparatus that has been described before were used. The first was the standard system described by Derkson, Pashley and Derkson (1986) to measure the hydraulic conductance (Lp) of dentine using crown segments. The system measured permeability by following the progress of a small air bubble within a 25 ~1 micropipette, 65 mm in length. The second was a modification of the original method which permitted more sensitive measurements consistent with evaporation. The modification incorporated a longer (100 mm) but much smaller diameter micropipette with a 3.17~1 total capacity (O.O317~l/mm x 100 mm) attached to appropriately sized tubing and, through a three-way connection, to a microsyringe (Fig. 1). The system was used with no external hydrostatic pressure and was therefore open at one end. The height between the end of the micropipette and the prepared dentine surface was carefully adjusted to nullify meniscus movement in the micropipette. The open end of the micropipette allowed an air-water interface to develop. Theoretically, the capillarity of the micropipette might produce a negative pressure which would slow or underestimate the rate of water evaporation. This was evaluated by measuring evaporation gravimetrically on an analytical balance in the absence of a pipette. No statistically significant differences were found, indicating that under the conditions employed in these experiments, the presence of a micropipette did not influence water evaporation. Procedure
After each crown segment had been attached to the Plexiglas square, it was then connected to the standard fluid filtration system, treated with 0.5 M EDTA at a pH of 7.4, and the maximum hydraulic conductance (Lp) of each sample determined. Distilled water was used in the system to avoid the reductions in hydraulic conductance that are known to occur after evaporation of phosphate-buffered saline solutions
and the apparatus loss.
used to measure
evaporative
(Pashley, Stewart and Galloway, 1984). The water contained fluorescein dye (0.2%) previously filtered through 0.2pm Millipore filters to remove particulate matter, in order more easily to visualize the movement of the tiny air bubble in the micropipette. All specimens were photographed at 12 x next to a millimetre rule to provide accurate measurement of the surface area of the dentine. This was determined by tracing the dentine on photographic prints on a digitizing tablet (Microcomp, Southern Micro Instruments, Inc., Atlanta, GA, U.S.A.). After the maximum Lp had been determined for each sample, the crown segment was sanded (Ecomet III, Buehler Ltd, Lake Bluff, IL, U.S.A.) with an 8 in Carbinet 320 grit disc (Buehler Ltd) for 5 s under a force of 500g at 108 rev/min to produce a standard smear layer (Tao, Pashley and Boyd, 1988). The hydraulic conductance of the smear layer-covered specimens was also determined. Finally, the crown segment was then attached to the more sensitive micropipette in the unpressurized system to measure evaporation. The teeth were not placed in a controlled temperature water bath after it had been determined that the evaporation-induced temperature changes were not consistent, were between 0.2 and 0.6”C and had no significant effect on the measurements made under the conditions of this experiment. The following procedures carried out at 25 k 2’C and 50 f 5% relative humidity: (1) Control or zero evaporation was measured while the dentine surface was covered with a drop of water. (2) Spontaneous evaporation was measured after removal of the drop of water by observing the bubble movement in the micropipette over a timed period. The smear layer was left in place. (3) An air syringe (Adec, Newberg, OR, U.S.A.) positioned at a 45” angle, 1 cm from the dentine surface was used to blow air for 1 min (147 cm’/s, 15 psi). The rate of evapo-
Evaporative water loss from human dentine
(4) (5) (6)
(7)
ration was measured during the air blast and this was repeated 5 times for each specimen. The air syringe was positioned a second time at a 90” angle and No. 3 repeated. The air syringe was positioned a third time at a 180” angle and No. 3 repeated. The smear layer was removed by etching the surface for I min with a 0.5 M EDTA solution (pH 7.4) and the above measurements at all 3 angles repeated. Control or zero evaporation was redetermined as in No. 1 above.
20 r
‘E15
i .E E I s e
10
Calculations
Four to eight replications of the measurement were made for each specimen at each step in the protocol. The means and variance of each measurement were then calculated and an analysis of variance of repeated measures was used to identify differences among the groups. The rates of evaporation (~1 min-’ cmm2) for each phase of the experiment were calculated along with the Lp values. The results were subjected to a one-way analysis of variance of repeated measures and Student’s r-test. RESULTS
The rates of spontaneous and air blast-induced evaporative water loss from the surfaces of 6 crown segments in the presence and absence of smear layers are shown in Table 1. The spontaneous rate of evaporation varied but ranged from 0.7 to 1.3 ~1 min-’ cmm2 in the presence of a smear layer from 0.6 to 1.O~1 min-’ cm-* in the absence of a smear layer. There were no consistent statistically significant differences between these rates in the presence or absence of smear layers (NS, Table 1).
cont&i -
+sL
450
900
moo-SL
+ Smear layer--
45’
90’
woo Control
- Smear layer-w
Fig. 2. Evaporative water loss before (+) and after (-) removal of smear layer and exposure to air blasts at different directions.
There was no evaporation from the dentine when the surface was covered with a drop of water (Fig. 2). Removal of the water drop was associated with a small but consistent spontaneous loss of water from the tooth (Table 1 and Fig. 2). Subjecting the dentine surfaces to air blasts produced a large increase in the rate of evaporative water loss even in the presence of a smear layer. There were no statistically significant differences among the rates of water evaporation obtained at the 3 angles of the air syringe, although the values were generally higher at 90”. Air blast-induced evaporation rates were always higher after removal of the smear layer (Table 1 and Fig. 2). There was no correlation between rates of spontaneous or air blast-induced evaporation
Table 1. Evaporative u,ater loss in presence (+) and absence (-) of smear layer before and during air blasts directed at different angles Evaporative water loss ($1 min-’ cm-l) Crown segment
Smear layer
1
+
_ 2
+
3
+
_ 4
+
5
+
6
+
-
-Spontaneous*
45”T
90”
180”
0.56 f 0.21 NS 0.78 + 0.09 0.71 _+0.10 NS 0.77 f 0.09 0.83 + 0.06 NS 0.87 + 0.07 0.71 * 0.13 NS 0.56 rf: 0.05 1.26f 0.03 $ 1.03* 0.03 1.00+0.04 NS 1.00+0.17
6.92 + 0.46 5 13.36 k 0.98 6.03 k 1.50 $ 11.40 + 0.32 10.93 k 0.69 NS 12.15 k 0.36 2.15 &-0.15 NS 3.10 f 0.55 4.76kO.15 § 11.17_+0.80 1.84 f 0.14 § 3.85 + 0.15
7.34 * 0.73
2.57 f 0.82
5
0
15.09 * 4.27 k 4 17.05; 11.26k § 17.19 f 2.11 + $ 3.49 + 4.48 f 9 18.41 f 2.00 + § 5.93 +
0.44 0.95
0.56 0.23
11.57 + 0.70 3.75 + 0.45 0 11.13kO.18 8.46 k 0.69 $ 11.94+0.14 1.11 kO.25 NS 3.28 f 0.90 3.68 & 0.23 0 16.86 + 1.12 2.03 f 0.1 I
0.24
5.72 & 0.17
0.32 0.82 0.68 0.19 0.27 0.39
5
*Eight measurements were averaged for each tooth when studying spontaneous evaporation. tFour measurements were averaged for each tooth when studying air-induced evaporation. $p < 0.05. &I < 0.01. NS = not statistically srgnificant (p > 0.05). AOB 35,7-c
LP (pi crnm2min-’ cmH,O-’ 8.89 x 10-j NS 1.09 x 10-l 1.98 x IO-’ § 1.05 x IO-’ 4.35 x 10-j 0 2.64* x 10-l 9.37 x IO-’ 5 3.67k x 1O-2 1.90* x IO-3 0 1.77+ x IO-’ 2.12 x 1010-3 0 1.57 x IO-’
H. E. G~ODIS et al.
526 and the hydraulic (Table 1).
conductance
of the specimens
DISCUSSION
We have demonstrated that evaporation of water does, indeed, occur at dentine surfaces under our test conditions. When a segment was covered with water by placing a large drop over the dentine surface, evaporation ceased: this was an important control; water was still evaporating from the surface of the water but not from the dentine. When the water drop was removed, the dentine surfaces were exposed to the ambient environment and water began leaving dentine as it changed from a liquid to a gaseous state, thereby allowing evaporation to resume. Theoretically, the capillary connected to the crown segment should have produced a braking action on evaporation due to the air-water interface of the meniscus in the pipette. However, when the experiments were repeated using weight changes to quantify evaporation in specimens with or without pipettes, the results were not statistically different. This may be due to the fact that all pipettes were filled with water before they were connected to the teeth. There was no correlation between the rate of water evaporation from each segment and that segment’s hydraulic conductance. Specimens with the highest hydraulic conductances did not necessarily have the highest evaporation rates, while a specimen with a very low hydraulic conductance had the highest evaporation rate (specimen No. 5, Table 1). This was true for specimens with and without smear layer, suggesting that the condition of the cut dentine surface and the Lp may have no effect on evaporation from that surface. It is more likely that the rate of evaporation would be more dependent on the ambient temperature, relative humidity, surface area and amount of convection of air. The large variation in evaporation rates between samples, even after controlling for dentine surface area and thickness, suggests that there is a great deal of biological variability between teeth in regard of water loss; this has been discussed elsewhere in greater detail (Pashley, 1990). As water evaporates from dentine, surface tension presumably holds in place the meniscus of fluid in the Table 2. Comparison
of evaporation
tubule orifice so that any water lost from dentine creates a slight negative hydrostatic pressure which pulls the same amount of fluid from the pulp chamber into the dentine. The mechanoreceptors in dentine may be sensitive to pressure (Matthews and Hughes, 1988) rather than to fluid flow. Water evaporation from dentine was reported by Brannstriim and his associates (Brannstriim and Astrom, 1964, 1972; Brannstrom, Linden and Astrom, 1967) as they marshalled evidence for the hydrodynamic theory of dentine sensitivity. They demonstrated that air blasts applied to cut dentine surfaces drew water from the pulp chamber. Our findings confirm those of Brannstriim and extend them by permitting quantification of spontaneous as well as air blast-induced evaporation. Previous experience of measuring pulpal tissue pressure (Pashley, Nelson and Pashley, 1981) and the hydraulic conductance of dentine (Reeder et al., 1978; Fogel et al., 1988) gave us an important perspective from which to interpret the rates of water evaporation. The rate of fluid filtration across dentine can be calculated as the product of the hydraulic conductance of the specimen and the pulpal tissue pressure expressed in centimetres of water. By assuming a pulpal tissue pressure of 30cm H,O (Pashley et al., 1981) we have calculated the amount of fluid filtration that would have occurred across each of the 6 specimens had we applied such a pressure, using the measured hydraulic conductances of each specimen (Table 2). Note that the measured rates of water evaporation were almost an order of magnitude larger than the calculated filtration rates and that evaporative water loss accounted for 67-95% of the total net water flux across dentine covered with a smear layer. The same type of comparisons were made using the data obtained from EDTA-etched dentine (minus smear layer values in Table 2). In the absence of a smear layer, evaporative water loss accounted for between 18 and 72% of the total fluid flux across the dentine. In the absence of the smear layer, filtration accounted for more of the total fluid flux in 3 out of the 6 specimens. These calculations were useful in demonstrating the large contribution made by evaporative water loss to the total
vs filtration
to total fluid flux across
dentine
Estimated Specimen No. 1 2 3 4 5 6 *Filtration pulpal
Smear layer
Evaporation (A) (~1 min-’ cm-‘)
Filtration* (B) 011 min-’ cmm2)
Total fluid flux (A + B) (PI min’ cmm2)
A as % of (A+B)
+ _ + + + + + -
0.56 0.78 0.71 0.77 0.83 0.87 0.71 0.56 1.26 1.03
0.27 0.30 0.06 3.15 0.13 0.79 0.28 1.10 0.06 0.53 0.06 4.71
0.83 1.08 0.77 3.92 0.96 1.66 0.99 1.66 1.32 1.56 1.06 5.71
67 72 92 20 86 52 72 34 95 66 94 18
I .oo 1.00
rates were calculated as the product pressure of 30cm of H,O.
of the measured
Lps in Table
1 and an assumed
Evaporative water loss from human dentine amount of fluid flow across dentine in the presence of a smear layer. If a clinician were to be operating a highspeed dental handpiece with an air-spray only, the evaporative water loss across dentine might be larger (and similar to our air-blast values) than might occur with an air-water spray, which would tend to minimize evaporative water loss from dentine. This would tend to cause more displacement of odontoblasts into dentinal tubules. Several histological studies of the effects of dry versus wet cutting have confirmed this notion (Langeland, 1959; Hamilton and Kramer, 1967). Cutiing under dry conditions would transiently maximize hydrodynamic stimuli until the dentine surfaces became dry and insensitive (Polhagen and Brlinnstrom, 1971; Johnson and Brannstrom, 1974). At that point, dentine appears to be less sensitive than normal until it is rehydrated or until the superficial part is removed by grinding (Johnson and Brannstrom, 1974). The hydrodynamic theory states that dentine sensitivity is due to movement of fluid contents within dentinal tubules which, in turn, deforms and activates mechanoreceptors. Current clinical research in dentine sensitivity is directed at correlating the number and degree assessments
of patency of tubules with quantitative of pain. The use of quantitative stimuli
such as tactile force (Clark, Al-Joburi and Chan, 1987; Minkoff and Axelrod, 1987) or graded cold water (Brough et al., 1985; Muzzin and Johnson, 1989) have been usef’ul in assessing pain. No one has devised a method of grading air blasts to provide continuously variable, quantitative stimuli. Our technique provides a simple, convenient method for quantifying evaporative water loss from dentine. As our experiments were conducted at 25°C they represent minimum values for spontaneous water evaporation relative to those that may be measured at body temperature. Acknowledgements-Tllis was supported, in part, by DE 06427 from the National Institute for Dental Research, by the Medical College of Georgia Dental Research Center and by a Pew Pretenure Fellowship Award to Harold E. Goodis.
REFERENCES Brannstrom M. and Astrom A. (1964) A study on the mechanisms of pain elicited from dentine. J. dent. Res. 43, 619625.
Brlnnstriim M. and Astrom A. (1972) The hydrodynamics of the dentine: its possible relationship to dentinal pain. IN. dent. J. 22, 219-227.
527
Briinnstrdm M. and Johnson G. (1978) The sensory mechanism in human dentin as revealed by evaporation and mechanical removal of dentin. J. dent. Res. 57, 49-53.
Brannstriim M., Linden L. A. and Astrom A. (1967) The hydrodynamics of the dental tubule and of pulp fluid. Curies Res. 1, 310-317. Brough K. M., Anderson D. M., Love J. and Overman P. R. (1985) The effectiveness of iontophoresis in reducing dentin hypersensitivity. J. Am. dent. Ass. 111. 761-764. Clark D. C.: Al-Joburi-W. and Chan E. C. S. (1987) The efficacy of a new dentifrice in treating dentin sensitivity: effect of sodium citrate and sodium fluoride as active ingredients. J. periodont. Res. 22, 89-93. Derkson G. D., Pashley D. H. and Derkson M. E. (1986) Microleakage measurement of selected restorative materials: a new in clitro method. J. prosthet. Dent. 56. 435-444.
Fogel H. M., Marshall F. J. and Pashley D. H. (1988) Effects of distance from the pulp and thickness on the hydraulic conductance of human radicular dentin. J. dent. Res. 67, 1381-1385. Hamilton A. I. and Kramer I. R. H. (1967) Cavity preparation with and without waterspray. Br. dent. J. 123, 281-285.
Johnson G. and BrLnnstrom M. (1974) The sensitivity of dentin changes in relation to conditions at exposed tubule operatures. Acta odont. stand. 32, 29-38. Langeland K. (1959) Histologic evaluation of pulp reactions to operative procedures. Oral Surg. 12, 1235-1248. Matthews B. and Hughes S. H. S. (1988) The ultrastructure and receptor transduction mechanisms of dentine. In: Progress in Brain Research (Edited by Hamann W. and Iggo A.) Vol. 74, pp. 69-76. Elsevier, New York. Minkoff S. and Axelrod S. (1987) Efficacv of strontium chloride in dental hypersensitivity. J. ‘Periodont. 58, 470474.
Muzzin K. B. and Johnson R. (1989) Effects of potassium oxalate on dentin hypersensitivity in uiuo. J. Periodont. 60, 151-158.
Pashley D. H. (1990) Dentin permeability: theory and practice. In: Experimental Endodontics (Edited by Spang_ _ berg L.). CRC-Press, Boca Raton, FL. Pashley D. H., Nelson R. and Pashley E. L. (1981) In uioo fluid movement across dentine in the doa. Archs oral Bial. 26, 707-7 10. Pashley D. H., Stewart F. P. and Galloway S. E. (1984) Effects of air-drving in vitro on human dentine nermeability. Archs oral bioT. 29, 379-383. Polhagen L. and Brlnnstriim M. (1971) The liquid movement in desiccated and rehydrated dentine in vitro. Acta odont. stand. 29, 95-102.
Reeder 0. W., Walton R. E., Livingston M. J. and Pashley D. H. (1978) Dentine nermeabilitv: Determinants of hydraulic conductance. i dent. Res.-57, 187-193. Tao L., Pashley D. H. and Boyd L. (1988) Effect of different types of smear layers on dentin and enamel shear bond strengths. Denf. Mater. 4, 208-216.
EVAPORATIVE
1990 Copyright
WATER
LOSS FROM IN VITRO
HUMAN
0003-9969/90 $3.00 + 0.00 % 1990 Pergarbn Press plc
DENTINE
H. E. GOODIS,’ L. TAO’ and D. H. PASHLEY’ ‘Departmentof Restorative Dentistry, University of California, San Francisco, CA 94143 and ‘Department of Oral Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A. (Accepted 30 January 1990) Summary-The rate of spontaneous evaporation of water from dentine was measured in extracted human teeth in vitro. !;pontaneous water loss was the same with or without a smear layer. When air was blown on the dentine, the rate of evaporation increased significantly. After removal of the smear layer, the air blast-induced evaporative loss was twice as great as before its removal. Thus, with a smear layer present, evaporation is the major route by which fluid is lost from dentine rather than by filtration of dentinal fluid. After smear layer removal, fluid filtration sometimes may exceed the spontaneous rate of fluid evaporation. Key words:
evaporation,
dentinal
fluid, dentine,
filtration,
dentine
sensitivity,
MATERIALS
IhTRODUCTION
Crown segment
During many dental procedures large amounts of dentine are exposed by removal of peripheral hard tissues. This provides the potential for movement of pulpal fluid to the surface. There are two mechanisms by which fluid so moves, convection and evaporation. The rat12of convection is the product of the pulpal tissue pressure and the hydraulic conductance of the dentine (Reeder et al., 1978; Fogel, Marshall and Pashley, 1988). Dentine covered with a smear layer has a relatively low hydraulic conductance. Even in the absence of a smear layer, the rate of fluid flow through dentinal tubules brought about by filtration of pulpal fluid down existing hydrostatic pressure gradients is usually too low to stimulate pulpal mechanoreceptors via hydrodynamic effects. H:owever, when such exposed dentine is subjected to a blast of air, water evaporates from it at a rate which can cause pain (Brannstrom and Astrom, 1964; Brannstriim and Johnson, 1978). Nothing is known about the relative rates of spontaneous evaporation from dentine in the presence or absence of smear layers or how much evaporation increases following air blasts. BrHnnstriim and Astriim (1964) demonstrated that air blasts to dentine cause a measurable amount of water evaporation from dentine. Their h vitro system was not sensitive enough to measure spontaneous evaporation, although in vivo allowing dentine to evaporate spontaneously to dryness caused pain (Brannstrom and Johnson, 1978). Our purpose was to develop a method for quantifying the evaporation of water from dentine surfaces in vitro, in the presence and absence of smear layers and in response to air blasts, and to compare those fluid fluxes with fluid filtration rates.
hydrodynamic
theory.
AND METHODS
preparation
Six extracted, unerupted third molars were used. Immediately after extraction, they were placed in an isotonic saline solution (phosphate-buffered saline) containing 0.2% sodium azide to prevent bacterial growth, and stored at 4°C for less than 1 month. Crown segments were prepared in the following manner. The occlusal surface of the tooth was attached to a plastic mount with an epoxy resin cement (Epoxy, Cole Palmer, Chicago, IL, U.S.A.). The mount was placed in a holding device attached to a cutting saw (Isomet, Buehler Ltd, Lake Bluff, IL, U.S.A.). Two parallel cuts were made with a diamond blade under water coolant. The first cut removed the roots near the cementumenamel junction and the second removed the cusps and the occlusal enamel (Fig. 1). This produced a crown segment which contained the top of the coronal pulp chamber and a flat occlusal surface of dentine with no enamel except around the periphery. Any pulpal tissue remaining was removed, with care taken not to touch the dentine within the pulp chamber. Plexiglas squares (2 x 2 x 0.5 cm) were prepared with holes drilled through their centres. A 2-cm 18 ga stainless-steel tube was passed through the hole to end flush with one flat surface and to extend past the opposite surface (Fig. 1). The crown segment was then attached to the Plexiglas square with a cyanoacrylate adhesive (Zapit, DVA, Yorba Linda, CA, U.S.A.). At the end of experiments, the crowns were sectioned longitudinally to permit measurement of the thickness of dentine remaining between the highest pulp horn and the cut surface. This value ranged from 0.5 to 0.7 mm (0.5 f 0.2 f f SD, N = 6). 523
524
H. E. GOODIS er al. Crown segment 100mm
long, 34
capacity
micropipette
Microsyringe
Fig.
1. Schematic
diagram
of the tooth
preparation water
Apparatus
Two versions of an apparatus that has been described before were used. The first was the standard system described by Derkson, Pashley and Derkson (1986) to measure the hydraulic conductance (Lp) of dentine using crown segments. The system measured permeability by following the progress of a small air bubble within a 25 ~1 micropipette, 65 mm in length. The second was a modification of the original method which permitted more sensitive measurements consistent with evaporation. The modification incorporated a longer (100 mm) but much smaller diameter micropipette with a 3.17~1 total capacity (O.O317~l/mm x 100 mm) attached to appropriately sized tubing and, through a three-way connection, to a microsyringe (Fig. 1). The system was used with no external hydrostatic pressure and was therefore open at one end. The height between the end of the micropipette and the prepared dentine surface was carefully adjusted to nullify meniscus movement in the micropipette. The open end of the micropipette allowed an air-water interface to develop. Theoretically, the capillarity of the micropipette might produce a negative pressure which would slow or underestimate the rate of water evaporation. This was evaluated by measuring evaporation gravimetrically on an analytical balance in the absence of a pipette. No statistically significant differences were found, indicating that under the conditions employed in these experiments, the presence of a micropipette did not influence water evaporation. Procedure
After each crown segment had been attached to the Plexiglas square, it was then connected to the standard fluid filtration system, treated with 0.5 M EDTA at a pH of 7.4, and the maximum hydraulic conductance (Lp) of each sample determined. Distilled water was used in the system to avoid the reductions in hydraulic conductance that are known to occur after evaporation of phosphate-buffered saline solutions
and the apparatus loss.
used to measure
evaporative
(Pashley, Stewart and Galloway, 1984). The water contained fluorescein dye (0.2%) previously filtered through 0.2pm Millipore filters to remove particulate matter, in order more easily to visualize the movement of the tiny air bubble in the micropipette. All specimens were photographed at 12 x next to a millimetre rule to provide accurate measurement of the surface area of the dentine. This was determined by tracing the dentine on photographic prints on a digitizing tablet (Microcomp, Southern Micro Instruments, Inc., Atlanta, GA, U.S.A.). After the maximum Lp had been determined for each sample, the crown segment was sanded (Ecomet III, Buehler Ltd, Lake Bluff, IL, U.S.A.) with an 8 in Carbinet 320 grit disc (Buehler Ltd) for 5 s under a force of 500g at 108 rev/min to produce a standard smear layer (Tao, Pashley and Boyd, 1988). The hydraulic conductance of the smear layer-covered specimens was also determined. Finally, the crown segment was then attached to the more sensitive micropipette in the unpressurized system to measure evaporation. The teeth were not placed in a controlled temperature water bath after it had been determined that the evaporation-induced temperature changes were not consistent, were between 0.2 and 0.6”C and had no significant effect on the measurements made under the conditions of this experiment. The following procedures carried out at 25 k 2’C and 50 f 5% relative humidity: (1) Control or zero evaporation was measured while the dentine surface was covered with a drop of water. (2) Spontaneous evaporation was measured after removal of the drop of water by observing the bubble movement in the micropipette over a timed period. The smear layer was left in place. (3) An air syringe (Adec, Newberg, OR, U.S.A.) positioned at a 45” angle, 1 cm from the dentine surface was used to blow air for 1 min (147 cm’/s, 15 psi). The rate of evapo-
Evaporative water loss from human dentine
(4) (5) (6)
(7)
ration was measured during the air blast and this was repeated 5 times for each specimen. The air syringe was positioned a second time at a 90” angle and No. 3 repeated. The air syringe was positioned a third time at a 180” angle and No. 3 repeated. The smear layer was removed by etching the surface for I min with a 0.5 M EDTA solution (pH 7.4) and the above measurements at all 3 angles repeated. Control or zero evaporation was redetermined as in No. 1 above.
20 r
‘E15
i .E E I s e
10
Calculations
Four to eight replications of the measurement were made for each specimen at each step in the protocol. The means and variance of each measurement were then calculated and an analysis of variance of repeated measures was used to identify differences among the groups. The rates of evaporation (~1 min-’ cmm2) for each phase of the experiment were calculated along with the Lp values. The results were subjected to a one-way analysis of variance of repeated measures and Student’s r-test. RESULTS
The rates of spontaneous and air blast-induced evaporative water loss from the surfaces of 6 crown segments in the presence and absence of smear layers are shown in Table 1. The spontaneous rate of evaporation varied but ranged from 0.7 to 1.3 ~1 min-’ cmm2 in the presence of a smear layer from 0.6 to 1.O~1 min-’ cm-* in the absence of a smear layer. There were no consistent statistically significant differences between these rates in the presence or absence of smear layers (NS, Table 1).
cont&i -
+sL
450
900
moo-SL
+ Smear layer--
45’
90’
woo Control
- Smear layer-w
Fig. 2. Evaporative water loss before (+) and after (-) removal of smear layer and exposure to air blasts at different directions.
There was no evaporation from the dentine when the surface was covered with a drop of water (Fig. 2). Removal of the water drop was associated with a small but consistent spontaneous loss of water from the tooth (Table 1 and Fig. 2). Subjecting the dentine surfaces to air blasts produced a large increase in the rate of evaporative water loss even in the presence of a smear layer. There were no statistically significant differences among the rates of water evaporation obtained at the 3 angles of the air syringe, although the values were generally higher at 90”. Air blast-induced evaporation rates were always higher after removal of the smear layer (Table 1 and Fig. 2). There was no correlation between rates of spontaneous or air blast-induced evaporation
Table 1. Evaporative u,ater loss in presence (+) and absence (-) of smear layer before and during air blasts directed at different angles Evaporative water loss ($1 min-’ cm-l) Crown segment
Smear layer
1
+
_ 2
+
3
+
_ 4
+
5
+
6
+
-
-Spontaneous*
45”T
90”
180”
0.56 f 0.21 NS 0.78 + 0.09 0.71 _+0.10 NS 0.77 f 0.09 0.83 + 0.06 NS 0.87 + 0.07 0.71 * 0.13 NS 0.56 rf: 0.05 1.26f 0.03 $ 1.03* 0.03 1.00+0.04 NS 1.00+0.17
6.92 + 0.46 5 13.36 k 0.98 6.03 k 1.50 $ 11.40 + 0.32 10.93 k 0.69 NS 12.15 k 0.36 2.15 &-0.15 NS 3.10 f 0.55 4.76kO.15 § 11.17_+0.80 1.84 f 0.14 § 3.85 + 0.15
7.34 * 0.73
2.57 f 0.82
5
0
15.09 * 4.27 k 4 17.05; 11.26k § 17.19 f 2.11 + $ 3.49 + 4.48 f 9 18.41 f 2.00 + § 5.93 +
0.44 0.95
0.56 0.23
11.57 + 0.70 3.75 + 0.45 0 11.13kO.18 8.46 k 0.69 $ 11.94+0.14 1.11 kO.25 NS 3.28 f 0.90 3.68 & 0.23 0 16.86 + 1.12 2.03 f 0.1 I
0.24
5.72 & 0.17
0.32 0.82 0.68 0.19 0.27 0.39
5
*Eight measurements were averaged for each tooth when studying spontaneous evaporation. tFour measurements were averaged for each tooth when studying air-induced evaporation. $p < 0.05. &I < 0.01. NS = not statistically srgnificant (p > 0.05). AOB 35,7-c
LP (pi crnm2min-’ cmH,O-’ 8.89 x 10-j NS 1.09 x 10-l 1.98 x IO-’ § 1.05 x IO-’ 4.35 x 10-j 0 2.64* x 10-l 9.37 x IO-’ 5 3.67k x 1O-2 1.90* x IO-3 0 1.77+ x IO-’ 2.12 x 1010-3 0 1.57 x IO-’
H. E. G~ODIS et al.
526 and the hydraulic (Table 1).
conductance
of the specimens
DISCUSSION
We have demonstrated that evaporation of water does, indeed, occur at dentine surfaces under our test conditions. When a segment was covered with water by placing a large drop over the dentine surface, evaporation ceased: this was an important control; water was still evaporating from the surface of the water but not from the dentine. When the water drop was removed, the dentine surfaces were exposed to the ambient environment and water began leaving dentine as it changed from a liquid to a gaseous state, thereby allowing evaporation to resume. Theoretically, the capillary connected to the crown segment should have produced a braking action on evaporation due to the air-water interface of the meniscus in the pipette. However, when the experiments were repeated using weight changes to quantify evaporation in specimens with or without pipettes, the results were not statistically different. This may be due to the fact that all pipettes were filled with water before they were connected to the teeth. There was no correlation between the rate of water evaporation from each segment and that segment’s hydraulic conductance. Specimens with the highest hydraulic conductances did not necessarily have the highest evaporation rates, while a specimen with a very low hydraulic conductance had the highest evaporation rate (specimen No. 5, Table 1). This was true for specimens with and without smear layer, suggesting that the condition of the cut dentine surface and the Lp may have no effect on evaporation from that surface. It is more likely that the rate of evaporation would be more dependent on the ambient temperature, relative humidity, surface area and amount of convection of air. The large variation in evaporation rates between samples, even after controlling for dentine surface area and thickness, suggests that there is a great deal of biological variability between teeth in regard of water loss; this has been discussed elsewhere in greater detail (Pashley, 1990). As water evaporates from dentine, surface tension presumably holds in place the meniscus of fluid in the Table 2. Comparison
of evaporation
tubule orifice so that any water lost from dentine creates a slight negative hydrostatic pressure which pulls the same amount of fluid from the pulp chamber into the dentine. The mechanoreceptors in dentine may be sensitive to pressure (Matthews and Hughes, 1988) rather than to fluid flow. Water evaporation from dentine was reported by Brannstriim and his associates (Brannstriim and Astrom, 1964, 1972; Brannstrom, Linden and Astrom, 1967) as they marshalled evidence for the hydrodynamic theory of dentine sensitivity. They demonstrated that air blasts applied to cut dentine surfaces drew water from the pulp chamber. Our findings confirm those of Brannstriim and extend them by permitting quantification of spontaneous as well as air blast-induced evaporation. Previous experience of measuring pulpal tissue pressure (Pashley, Nelson and Pashley, 1981) and the hydraulic conductance of dentine (Reeder et al., 1978; Fogel et al., 1988) gave us an important perspective from which to interpret the rates of water evaporation. The rate of fluid filtration across dentine can be calculated as the product of the hydraulic conductance of the specimen and the pulpal tissue pressure expressed in centimetres of water. By assuming a pulpal tissue pressure of 30cm H,O (Pashley et al., 1981) we have calculated the amount of fluid filtration that would have occurred across each of the 6 specimens had we applied such a pressure, using the measured hydraulic conductances of each specimen (Table 2). Note that the measured rates of water evaporation were almost an order of magnitude larger than the calculated filtration rates and that evaporative water loss accounted for 67-95% of the total net water flux across dentine covered with a smear layer. The same type of comparisons were made using the data obtained from EDTA-etched dentine (minus smear layer values in Table 2). In the absence of a smear layer, evaporative water loss accounted for between 18 and 72% of the total fluid flux across the dentine. In the absence of the smear layer, filtration accounted for more of the total fluid flux in 3 out of the 6 specimens. These calculations were useful in demonstrating the large contribution made by evaporative water loss to the total
vs filtration
to total fluid flux across
dentine
Estimated Specimen No. 1 2 3 4 5 6 *Filtration pulpal
Smear layer
Evaporation (A) (~1 min-’ cm-‘)
Filtration* (B) 011 min-’ cmm2)
Total fluid flux (A + B) (PI min’ cmm2)
A as % of (A+B)
+ _ + + + + + -
0.56 0.78 0.71 0.77 0.83 0.87 0.71 0.56 1.26 1.03
0.27 0.30 0.06 3.15 0.13 0.79 0.28 1.10 0.06 0.53 0.06 4.71
0.83 1.08 0.77 3.92 0.96 1.66 0.99 1.66 1.32 1.56 1.06 5.71
67 72 92 20 86 52 72 34 95 66 94 18
I .oo 1.00
rates were calculated as the product pressure of 30cm of H,O.
of the measured
Lps in Table
1 and an assumed
Evaporative water loss from human dentine amount of fluid flow across dentine in the presence of a smear layer. If a clinician were to be operating a highspeed dental handpiece with an air-spray only, the evaporative water loss across dentine might be larger (and similar to our air-blast values) than might occur with an air-water spray, which would tend to minimize evaporative water loss from dentine. This would tend to cause more displacement of odontoblasts into dentinal tubules. Several histological studies of the effects of dry versus wet cutting have confirmed this notion (Langeland, 1959; Hamilton and Kramer, 1967). Cutiing under dry conditions would transiently maximize hydrodynamic stimuli until the dentine surfaces became dry and insensitive (Polhagen and Brlinnstrom, 1971; Johnson and Brannstrom, 1974). At that point, dentine appears to be less sensitive than normal until it is rehydrated or until the superficial part is removed by grinding (Johnson and Brannstrom, 1974). The hydrodynamic theory states that dentine sensitivity is due to movement of fluid contents within dentinal tubules which, in turn, deforms and activates mechanoreceptors. Current clinical research in dentine sensitivity is directed at correlating the number and degree assessments
of patency of tubules with quantitative of pain. The use of quantitative stimuli
such as tactile force (Clark, Al-Joburi and Chan, 1987; Minkoff and Axelrod, 1987) or graded cold water (Brough et al., 1985; Muzzin and Johnson, 1989) have been usef’ul in assessing pain. No one has devised a method of grading air blasts to provide continuously variable, quantitative stimuli. Our technique provides a simple, convenient method for quantifying evaporative water loss from dentine. As our experiments were conducted at 25°C they represent minimum values for spontaneous water evaporation relative to those that may be measured at body temperature. Acknowledgements-Tllis was supported, in part, by DE 06427 from the National Institute for Dental Research, by the Medical College of Georgia Dental Research Center and by a Pew Pretenure Fellowship Award to Harold E. Goodis.
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Brlnnstriim M. and Astrom A. (1972) The hydrodynamics of the dentine: its possible relationship to dentinal pain. IN. dent. J. 22, 219-227.
527
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