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Effects of air-drying in vitro on human dentine permeability
Archs oral Biol. Vol. 29, No. 5. pp. 379-383. Printed in Great Britain. All rights reserved
EFFECTS
1984 Copyright
OF AIR-DRYING IN VITRO DENTINE PERMEABILITY
0003.9969184 $3.00 + 0.00 :c 1984 Pergamon Press Ltd
ON HUMAN
D. H. PASHLEY, F. P. STEWART and S. E. GALLOWAY Departments of Oral Biology and Physiology, Medical College of Georgia, Augusta,
GA 30912, U.S.A.
Summary-The effects of evaporation produced by air blasts of 0, 0.5, 2 or 5 mitt to dentine in vitro were evaluated by measuring dentine hydraulic conductance before and after each trial. When the tubules were tilled with water, even prolonged evaporation had no effect on dentine permeability. Tubules filled with physiological salt solution produced a time-dependent decrease in dentine permeability. Tubules filled with 1.5 per cent albumin in water gave the largest reductions in dentine permeability. These effects were more marked in unc tched as opposed to acid-etched dentine. The results suggest that part of the reduction in dentine sensitivity produced clinically by prolonged air blasts may be due to precipitation of organic and inorganic constituents of dentinal fluid at the surface.
INTRODUCTION
If human dentine is, dried with a stream of air for 5 min, it remains insensitive to painful stimuli as long as it is kept dry (Brlnnstriim, 1960). According to some workers, drying results in aspiration of odontoblast nuclei into the tubules. Some say the presence of aspirated organelles in the tubules (Cotton, 1967; Brannstrom, 1968; F’urseth and Mjor, 1969; Polhagen and Brinnstriim, 1971; Johnson and Brannstriim, 1974; Shiego and Saito, 1978; Lilja, Nordenvall and Brannstrbm, 1982) restricts the movement of fluid within the tubules and so decrease dentine sensitivity (Brannstrom, Linden and Astrom, 1967). Alternatively, prolonged air blasts could cause evaporation of water which would raise the concentration of organic and inorganic constituents of dentinal fluid so that they might precipitate in and on the tubule lumina. This would cause the same result, i.e. a decrease in fluid flow and, hence, in dentine sensitivity. In favour of the precipitation explanation is the observation that wetting previously dried, insensitive dentine restores its sensitivity, presumably by dissolving the precipitated material (Brannstrom, 1960). Such re-hyd;-ation does not reverse the aspiration of the odc’ntoblast nuclei (Hamilton and Kramer, 1967). Br.iinnstriim (1960) concluded that while aspiration of odontoblasts may contribute to reductions in sensitivity after dehydration of dentine, much of it was due to mechanical blockage of the orifices of dentinal tubules with salts and organic substances (Polhagen and Brannstriim, 1971). The use of prolonged air blasts reproducibly to aspirate odontoblast nuclei into the tubules is a model that has been used by many investigators. Although clinical e:#timates of dentine sensitivity have been attempted before and after such aspiration, no quantitative measures of fluid movement have been made in tiivo, before and after air blast-induced aspiration of odortoblasts. As this procedure also evaporates water from dentinal fluid and apparently causes precipitation of substances on the surface, a systematic study of the problem requires that the two phenomena (i.e. aspiration of odontoblast nuclei versus evaporative precipitation) be evaluated sepa379
rately to determine their contribution to the overall end result of dentine dehydration. The purpose of this experiment was to evaluate the ease with which fluid can move across human dentine in vitro, when the tubules were filled with water, physiological saline, a water solution of albumin or albumin in physiological saline, before and after acid-etching (i.e. in the presence or absence of the smear layer), in response to prolonged air blasts of 0.5, 2 or 5 min duration.
MATERIALS
AND METHODS
(A) Discs and chamber Discs of dentine, varying in thickness between 0.5 and 0.8 mm, prepared from human, unerupted third molars as previously described (Reeder et al., 1978) were placed in a split-chamber device modified so that the occlusal side of the disc was exposed to the atmosphere, with the pulp side confined in a chamber closed except for a single port which was connected to a pressure reservoir and microsyringe (Fig. 1). Double rubber 0 rings clamped on either side of the disc limited the exposed surface area to 0.282 cm’. All fluids used were prefiltered through 0.2 pm Millipore filters (Millipore Corp., Bedford, MA 01730, U.S.A.). Fluid movement was always in the pulp to enamel direction under a constant hydrostatic pressure of 30 cm H20 which simulates normal pulp tissuepressure (Stenvik, Iversen and Mjiir, 1972; Pashley, Nelson and Pashley, 1981). All discs were acid-etched for 2 min with 6 per cent citric acid on the pulp side so that only one smear layer was present (i.e. that on the enamel side) unless otherwise specified. (B) Measurement of hydraulic conductance 6,) A micropipette (5 ~1, Microcap, Fisher Scientific, Pittsburgh, PA 15219, U.S.A.) was introduced between the fluid reservoir and the chamber (Fig. 1). By following the progress of a small air bubble placed in the micropipette, the rate at which fluid moved across the dentine disc could be calculated. As the surface area, time, fluid flow and hydrostatic pressure gradient were all known, the data were expressed as
D. H.
380
hydraulic
conductances
PASHLEY
(L,), where:
L, = - J” AAPt
J, = fluid flow, in ~1; A = dentine surface area, in cm’; AP = hydrostatic pressure gradient, in cm H,O; t = time, in min; L, = hydraulic conductance, in ~1 cm-’ min-’ cm H,O-‘. Due to the extreme differences in L, obtained in unetched versus acid-etched dentine (Pashley, Michelich and Kehl, 1981) and to normalize the data for individual variations between discs, all data were expressed as percentage changes in ep, using the control values obtained before evaporation trials (i.e. airblasts) as 100 per cent. Thus, each disc served as its own control. Statistical differences were evaluated using the unpaired Student’s t-test. (C) Experimental
design
The fluid reservoir, polyethylene tubing, microsyringe and the lower portion of the split-chamber device were filled with either: (a) distilled water; (b) phosphate-buffered saline (Dulbecco PBS, Grand Island Biological Company, Grand Island, New York, U.S.A.); (c) 1.5 per cent bovine serum albumin, Fraction V (Grand Islands Biological Company, Cat. No. 526) in water or (d) 1.5 per cent bovine serum albumin, dissolved in phosphate-buffered saline. At least four discs were used in each experiment.
lb II (Hydrostatic Pressure)
i? E u
Dentine
Disc
w
et ul.
1. The first set of experiments was performed on discs which remained unetched on their enamel surfaces (i.e. with an intact smear layer on the enamel side of the dentine). The reservoir, tubing and chambers were filled with distilled water and control measurements of the hydraulic conductance (L,) of the discs were made using a hydrostatic head of 30 cm H,O pressure. Next, with the 30 cm H,O pressure still being applied to the pulp side of the disc, a continuous air blast of 0.5 min was applied to the open, enamel side of the dentine disc using a standard dental air syringe at a distance of 4 in. from the disc. Three more measurements of hydraulic conductance were made (10 min each for unetched discs) following the 0.5-min air blast. The protocol was repeated using a 2-min air blast. Hydraulic conductances were remeasured; then the discs were subjected to a 5 min continuous air blast. After re-determining the hydraulic conductances, these discs were discarded and new discs were used for the second series of experiments. 2. The second series of experiments were done exactly as above except that the reservoir, tubing and chamber were filled with Dulbecco phosphatebuffered saline (PBS). 3. The third set of experiments on new discs was done exactly as in set 1 except that the reservoir, tubing and chambers were filled with 1.5 per cent albumin dissolved in water. 4. The fourth set of experiments was done as in set 1 except that reservoir, tubing and chamber were filled with 1.5 per cent albumin dissolved in PBS instead of distilled water. 5. The entire set of experiments was repeated on discs that were acid-etched on both surfaces for 2 min with 6 per cent citric acid. These discs were termed acid-etched discs and were carried through all of the same experimental protocol except that, after the last measurements of hydraulic conductance were made following the 5 min air blast, the enamel side of each dentine disc was re-acid-etched with 6 per cent citric acid for 2 min and an additional set of measurements was made.
I RESULTS
Microsyringe
TO Strip Chart Recorder ---c
Fig. 1. Diagram of the apparatus used to measure the hydraulic conductance of dentine, in vitro. The movement of the air bubble toward the chamber represents the rate of fluid filtering across the dentine. The microsyringe permitted adjustment of the position of the air bubble between measurements.
Figure 2 (bottom) represents the data obtained in unetched discs (i.e. those with intact smear layer on the enamel side). There were no changes in L, when dentine filled with distilled water was subjected to air blasts for up to 5 min. When the tubules were filled with phosphate-buffered saline (PBS), exposure to 0.5-min air blasts reduced dentine hydraulic conductance to 79 per cent of the control value (p < 0.025). More prolonged evaporation produced progressively larger reductions in L, (to 72 and 69 per cent for 2 and 5 min, respectively, p < 0.005). Replacing the PBS with a protein solution (1.5 g per cent albumin) in water gave even larger reductions which were highly significant @ < 0.005) at every time period. When 1.5 g per cent albumin dissolved in PBS was used, similar results were obtained (i.e. there was a progressive decrease in L, with increasing time of evaporation). There were no statistically-significant differences between subgroups at the same times (i.e.
Effects of air-drying
on dentine
381
permeability
Etched
Unetched
co52
5
co5
2
5
co.5 15per
PBS
HP
2
co5
5
cent Albumm
15per
in H,O
2
5
cent AlbumIn In PBS
Fig. 2. Bar graphs of the percentage change in dentine L, in etched (top) or unetched (bottom) dentine, in oitro. C designates control values and 0.5, 2 and 5 indicate the duration of prolonged air blasts. The heights of the bars indicate the mean values and the vertical lines above each bar the magnitude of the standard error cf the mean of five discs in each group; open circle, indicates that the values are significantly different from controls at the p < 0.01 level; closed circle at the p < 0.025 and closed triangle at the p < 0.005 levels. PBS = phosphate-buffered saline.
Etched U netched
o--
4
-.
00
.* 0
10
y=-370x10-‘x +3.20 r=-073p c 0.01 y=14ox10-‘x+1 14 r=-043p < 001
. . 20
30
40
Percentage
50 change
60
70
80
90
In L,
Fig. 3. Fluid-filtration rate on the ordinate is plotted against percentage change in cp on the abscissa. The open circles indicate the data points obtained with acid-etched dentine and the sohd circles designate data obtained from unetched dentine.
382
D. H. PASHLEY et al
between PBS after 5 mm versus 1.5 per cent albumin in PBS at 5min). When the experiments were repeated on dentine which had been acid-etched on both sides, the results were similar (Fig. 2 top) to those in the presence of a smear layer, although only the 5-min values were statistically different from control values (p < 0.025). Generally, the reductions in L, were less in the acid-etched groups than the unetched groups although the differences did not reach statistical significance. Re-acid-etching the acid-etched dentine after 5-min air blasts restored the L, back to control values (not shown). The smaller decrease in Lp seen in acid-etched dentine was due to the solubtlizing effect of the higher rates of fluid flow seen in acidetched dentine (Fig. 3). The inverse relationship between dentine filtration rate and the fall in dentine L, (Fig. 3) suggests that the effect of evaporation may have been as great in both unetched and acid-etched dentine but that the putative precipitate was more quickly dissolved in the acid-etched group (i.e. the group that had a high fluid flow). DISCUSSION
By measuring changes in fluid filtration rates across dentine discs devoid of odontoblasts, in response to different evaporation time periods, the effects of evaporation on dentinal fluid were separated from those that produce aspiration of odontoblasts, in uiuo. The results implicate the presence of both inorganic salts and protein as responsible for up to a 50per cent reduction in dentine hydraulic conductance (Fig. 2). When dentine was filled with distilled water, there were no changes in dentine hydraulic conductance (L,) in either unetched or acid-etched dentine in response to air blasts of up to 5 min. Thus, air bubbles or air-locks in the tubules could not be responsible for decreasing dentine permeability. Presumably, under these conditions, the same amount of water was evaporating from these discs as was evaporating from subsequently studied discs; there was no change in L, because of evaporation of water from water does not change its composition. However, when inorganic salt solutions were used (PBS), evaporation of water presumably led to an increase in the concentration of the remaining salts, leading to crystalization or precipitation of unknown salts when their components exceeded their respective solubility products. More evaporation of water would occur at 5 than at 0.5 min, hence the larger reduction in L, after 5 min in both acid-etched and unetched dentine (Fig. 2). Dentinal-fluid protein concentrations are approx. 25 per cent that of plasma (von Kreudenstein, 1955; Haldi, Wynn and Culpepper, 1961; Pashley et al., 1981), and dog-plasma total protein is approx. 6 g per cent; thus, an albumin concentration of 1.5 g per cent simulates the protein concentration of dentinal fluid collected in vivo. In unetched dentine, 1.5 g per cent albumin in water produced reductions in L, at all time periods (p < O.OOS), presumably due to the evaporation of water producing a local increase in albumin concentration to the point where it exceeded its solubility in water and formed a precipitate on or in the smear layer.
Surprisingly, when the same concentration of albumin was dissolved in PBS rather than water, the result was not additive of the two separate effects but was similar to that obtained from either alone. Thus, at each time period there was a progressive decrease in L, (p < 0.005). Although the results of albumin plus PBS were not statistically different from albumin alone, there was a tendency at every time period for the reductions in L, for the albumin plus PBS to be less than those obtained with albumin alone. This may be due to the well-known increase salts produce on protein solubility (Mahler and Cordes, 1971). Thus, the simultaneous rise in salt and protein concentrations may facilitate higher protein solubilities than would be possible in water solutions of albumin. The lower reduction in L, in acid-etched dentine relative to that found in unetched dentine under similar conditions (compare Figs 2 versus 3), is due to a dependence of the decrease in L, on the rate of fluid flow (Fig. 3). Thus. the higher the flow rate, the lower was the decrease in L,. Acid etching removes the smear layer from dentine and permits fluids to flow through the tubules faster than unetched dentine, presumably redissolving both organic and inorganic precipitates. The lower filtration rates obtained in unetched dentine with an intact smear layer would not dissolve the precipitates as readily. Finally, when acid-etched dentine which had shown a large fall in L, was re-etched the L, was restored back to control values (not shown). This suggests that the material occluding dentine, whether organic or inorganic, is acid-labile and is present on or near the dentine surface. Our findings accord with Brannstrbm’s (1960) clinical observations, that prolonged air blasts decrease dentine sensitivity. The hydrodynamic theory of dentine sensitivity (Brlnnstrom et al., 1967) predicts that anything that decreases dentine fluid movement (i.e. dentine Lr) would decrease dentine sensitivity. We have shown that prolonged air blasts do decrease dentine .L,. If odontoblast nuclei were aspirated into the tubules, further reductions in L, would be expected. Acknowledgements-We thank Mrs Shirley Johnston for her secretarial assistance. This work was supported, in part. by NIDR Grant DE 06427. REFERENCES
Brlnnstrlim M. (1960) Dentinal and pulpal response II. Application of an airstream to exposed dentine. Short observation period. Acta odont. sand. 18, 17-28. Brannstriim M., Linden L. A. and Astrom A. (1967) The hydrodynamics of the dental tubule and of pulp fluid. Caries Rrs. 1, 310-317. Brannstriim M. (1968) The effect of dentine desiccation and aspirated odontoblasts on the pulp. d. pros/h. Dmr. 20, 165-171. Cotton W. R. (1967) Pulp response to an airstream directed into human cavity preparations. Oral Surg. 24, 78-88. Furseth R. and Mjiir I. (1969) Electron microscopy of human coronal dentine. A methodological study with emphasis on the “aspiration” of odontoblast nuclei. Acra odont. stand. 27, 577-593. Haldi J., Wynn W. and Culpepper W. D. (1961) Dental pulp fluid, I. Relationship between dental pulp fluid and blood plasma in protein, glucose and inorganic element content. Archs oral Biol. 3, 201-206.
Effects of air-drying
on dentine
Haldi J. and Wynn W. (1963) Protein fractions of the blood plasma and dental pulp fluid of the dog. J. dent. Res. 42, 1217-1221. Hamilton I. and Kramer I. R. H. (1967) Cavity preparation with and without water spray. Br. dent. J. 123, 281-285. Johnson G. and Brinnstrom M. (1974) The sensitivity of dentine. Changes in relation to conditions at exposed tubule apertures. Acta odont. stand. 32, 29-38. von Kreudenstein S. (1955) Dentinstoffwechselstudien I. Mitteilung iiber den ‘Dentin Liquor. Dr. zahniirtz. Z. 10, 473-476. Lilja J., Nordenvall K. J. and Brlnnstrom M. (1982) Dentin sensitivity, odontoblnsts and nerves under desiccated or infected experimental cavities. SW&. dent. J. 6, 93-103. Mahler H. R. and Cordes E. H. (1971) Bioloaical Chemistrv. 2nd edn, p. 99. Harper & Row, New York. Pashley D. H., Michelich V. and Kehl T. (1981) Dentin permeability: effect of smear layer removal. J. prosth. Dent. 46, 531-537.
permeability
383
Pashley D. H., Nelson R. and Pashley E. L. (1981) Fluid movement across dentine in the dog, in oiuo. Archs oral Biol. 26, 707-710. Pashley D. H., Nelson R., Williams E. C. and Kepler E. E. (1981) Use of dentine-Ruid protein concentrations to measure capillary reflection coefficients in dogs. Archs oral Biol. 26, 703-706. Polhagen L. and Brannstriim M. (1971) The liquid movement in desiccated and rehydrated dentine in vitro. Acta odont. stand. 29, 95-102. Reeder 0. W., Walton E. E., Livingston M. J. and Pashley D. H. (1978) Dentin permeability: determinants of hydraulic conductance. J. dent. Res. 51, 187-193. Shiego E. and Saito T. (1978) Electron microscopy of cells disnlaced into the dentinal tubules due to drvI cavitv_ preparation. J. oral Path. I, 326-335. Stenvik A., Iversen J. and Mjor I. (1972) Tissue pressure and histology of normal and inflamed tooth pulps in macaque monkeys. Archs oral Bioi. 17, 1501- 151 I.
EFFECTS
1984 Copyright
OF AIR-DRYING IN VITRO DENTINE PERMEABILITY
0003.9969184 $3.00 + 0.00 :c 1984 Pergamon Press Ltd
ON HUMAN
D. H. PASHLEY, F. P. STEWART and S. E. GALLOWAY Departments of Oral Biology and Physiology, Medical College of Georgia, Augusta,
GA 30912, U.S.A.
Summary-The effects of evaporation produced by air blasts of 0, 0.5, 2 or 5 mitt to dentine in vitro were evaluated by measuring dentine hydraulic conductance before and after each trial. When the tubules were tilled with water, even prolonged evaporation had no effect on dentine permeability. Tubules filled with physiological salt solution produced a time-dependent decrease in dentine permeability. Tubules filled with 1.5 per cent albumin in water gave the largest reductions in dentine permeability. These effects were more marked in unc tched as opposed to acid-etched dentine. The results suggest that part of the reduction in dentine sensitivity produced clinically by prolonged air blasts may be due to precipitation of organic and inorganic constituents of dentinal fluid at the surface.
INTRODUCTION
If human dentine is, dried with a stream of air for 5 min, it remains insensitive to painful stimuli as long as it is kept dry (Brlnnstriim, 1960). According to some workers, drying results in aspiration of odontoblast nuclei into the tubules. Some say the presence of aspirated organelles in the tubules (Cotton, 1967; Brannstrom, 1968; F’urseth and Mjor, 1969; Polhagen and Brinnstriim, 1971; Johnson and Brannstriim, 1974; Shiego and Saito, 1978; Lilja, Nordenvall and Brannstrbm, 1982) restricts the movement of fluid within the tubules and so decrease dentine sensitivity (Brannstrom, Linden and Astrom, 1967). Alternatively, prolonged air blasts could cause evaporation of water which would raise the concentration of organic and inorganic constituents of dentinal fluid so that they might precipitate in and on the tubule lumina. This would cause the same result, i.e. a decrease in fluid flow and, hence, in dentine sensitivity. In favour of the precipitation explanation is the observation that wetting previously dried, insensitive dentine restores its sensitivity, presumably by dissolving the precipitated material (Brannstrom, 1960). Such re-hyd;-ation does not reverse the aspiration of the odc’ntoblast nuclei (Hamilton and Kramer, 1967). Br.iinnstriim (1960) concluded that while aspiration of odontoblasts may contribute to reductions in sensitivity after dehydration of dentine, much of it was due to mechanical blockage of the orifices of dentinal tubules with salts and organic substances (Polhagen and Brannstriim, 1971). The use of prolonged air blasts reproducibly to aspirate odontoblast nuclei into the tubules is a model that has been used by many investigators. Although clinical e:#timates of dentine sensitivity have been attempted before and after such aspiration, no quantitative measures of fluid movement have been made in tiivo, before and after air blast-induced aspiration of odortoblasts. As this procedure also evaporates water from dentinal fluid and apparently causes precipitation of substances on the surface, a systematic study of the problem requires that the two phenomena (i.e. aspiration of odontoblast nuclei versus evaporative precipitation) be evaluated sepa379
rately to determine their contribution to the overall end result of dentine dehydration. The purpose of this experiment was to evaluate the ease with which fluid can move across human dentine in vitro, when the tubules were filled with water, physiological saline, a water solution of albumin or albumin in physiological saline, before and after acid-etching (i.e. in the presence or absence of the smear layer), in response to prolonged air blasts of 0.5, 2 or 5 min duration.
MATERIALS
AND METHODS
(A) Discs and chamber Discs of dentine, varying in thickness between 0.5 and 0.8 mm, prepared from human, unerupted third molars as previously described (Reeder et al., 1978) were placed in a split-chamber device modified so that the occlusal side of the disc was exposed to the atmosphere, with the pulp side confined in a chamber closed except for a single port which was connected to a pressure reservoir and microsyringe (Fig. 1). Double rubber 0 rings clamped on either side of the disc limited the exposed surface area to 0.282 cm’. All fluids used were prefiltered through 0.2 pm Millipore filters (Millipore Corp., Bedford, MA 01730, U.S.A.). Fluid movement was always in the pulp to enamel direction under a constant hydrostatic pressure of 30 cm H20 which simulates normal pulp tissuepressure (Stenvik, Iversen and Mjiir, 1972; Pashley, Nelson and Pashley, 1981). All discs were acid-etched for 2 min with 6 per cent citric acid on the pulp side so that only one smear layer was present (i.e. that on the enamel side) unless otherwise specified. (B) Measurement of hydraulic conductance 6,) A micropipette (5 ~1, Microcap, Fisher Scientific, Pittsburgh, PA 15219, U.S.A.) was introduced between the fluid reservoir and the chamber (Fig. 1). By following the progress of a small air bubble placed in the micropipette, the rate at which fluid moved across the dentine disc could be calculated. As the surface area, time, fluid flow and hydrostatic pressure gradient were all known, the data were expressed as
D. H.
380
hydraulic
conductances
PASHLEY
(L,), where:
L, = - J” AAPt
J, = fluid flow, in ~1; A = dentine surface area, in cm’; AP = hydrostatic pressure gradient, in cm H,O; t = time, in min; L, = hydraulic conductance, in ~1 cm-’ min-’ cm H,O-‘. Due to the extreme differences in L, obtained in unetched versus acid-etched dentine (Pashley, Michelich and Kehl, 1981) and to normalize the data for individual variations between discs, all data were expressed as percentage changes in ep, using the control values obtained before evaporation trials (i.e. airblasts) as 100 per cent. Thus, each disc served as its own control. Statistical differences were evaluated using the unpaired Student’s t-test. (C) Experimental
design
The fluid reservoir, polyethylene tubing, microsyringe and the lower portion of the split-chamber device were filled with either: (a) distilled water; (b) phosphate-buffered saline (Dulbecco PBS, Grand Island Biological Company, Grand Island, New York, U.S.A.); (c) 1.5 per cent bovine serum albumin, Fraction V (Grand Islands Biological Company, Cat. No. 526) in water or (d) 1.5 per cent bovine serum albumin, dissolved in phosphate-buffered saline. At least four discs were used in each experiment.
lb II (Hydrostatic Pressure)
i? E u
Dentine
Disc
w
et ul.
1. The first set of experiments was performed on discs which remained unetched on their enamel surfaces (i.e. with an intact smear layer on the enamel side of the dentine). The reservoir, tubing and chambers were filled with distilled water and control measurements of the hydraulic conductance (L,) of the discs were made using a hydrostatic head of 30 cm H,O pressure. Next, with the 30 cm H,O pressure still being applied to the pulp side of the disc, a continuous air blast of 0.5 min was applied to the open, enamel side of the dentine disc using a standard dental air syringe at a distance of 4 in. from the disc. Three more measurements of hydraulic conductance were made (10 min each for unetched discs) following the 0.5-min air blast. The protocol was repeated using a 2-min air blast. Hydraulic conductances were remeasured; then the discs were subjected to a 5 min continuous air blast. After re-determining the hydraulic conductances, these discs were discarded and new discs were used for the second series of experiments. 2. The second series of experiments were done exactly as above except that the reservoir, tubing and chamber were filled with Dulbecco phosphatebuffered saline (PBS). 3. The third set of experiments on new discs was done exactly as in set 1 except that the reservoir, tubing and chambers were filled with 1.5 per cent albumin dissolved in water. 4. The fourth set of experiments was done as in set 1 except that reservoir, tubing and chamber were filled with 1.5 per cent albumin dissolved in PBS instead of distilled water. 5. The entire set of experiments was repeated on discs that were acid-etched on both surfaces for 2 min with 6 per cent citric acid. These discs were termed acid-etched discs and were carried through all of the same experimental protocol except that, after the last measurements of hydraulic conductance were made following the 5 min air blast, the enamel side of each dentine disc was re-acid-etched with 6 per cent citric acid for 2 min and an additional set of measurements was made.
I RESULTS
Microsyringe
TO Strip Chart Recorder ---c
Fig. 1. Diagram of the apparatus used to measure the hydraulic conductance of dentine, in vitro. The movement of the air bubble toward the chamber represents the rate of fluid filtering across the dentine. The microsyringe permitted adjustment of the position of the air bubble between measurements.
Figure 2 (bottom) represents the data obtained in unetched discs (i.e. those with intact smear layer on the enamel side). There were no changes in L, when dentine filled with distilled water was subjected to air blasts for up to 5 min. When the tubules were filled with phosphate-buffered saline (PBS), exposure to 0.5-min air blasts reduced dentine hydraulic conductance to 79 per cent of the control value (p < 0.025). More prolonged evaporation produced progressively larger reductions in L, (to 72 and 69 per cent for 2 and 5 min, respectively, p < 0.005). Replacing the PBS with a protein solution (1.5 g per cent albumin) in water gave even larger reductions which were highly significant @ < 0.005) at every time period. When 1.5 g per cent albumin dissolved in PBS was used, similar results were obtained (i.e. there was a progressive decrease in L, with increasing time of evaporation). There were no statistically-significant differences between subgroups at the same times (i.e.
Effects of air-drying
on dentine
381
permeability
Etched
Unetched
co52
5
co5
2
5
co.5 15per
PBS
HP
2
co5
5
cent Albumm
15per
in H,O
2
5
cent AlbumIn In PBS
Fig. 2. Bar graphs of the percentage change in dentine L, in etched (top) or unetched (bottom) dentine, in oitro. C designates control values and 0.5, 2 and 5 indicate the duration of prolonged air blasts. The heights of the bars indicate the mean values and the vertical lines above each bar the magnitude of the standard error cf the mean of five discs in each group; open circle, indicates that the values are significantly different from controls at the p < 0.01 level; closed circle at the p < 0.025 and closed triangle at the p < 0.005 levels. PBS = phosphate-buffered saline.
Etched U netched
o--
4
-.
00
.* 0
10
y=-370x10-‘x +3.20 r=-073p c 0.01 y=14ox10-‘x+1 14 r=-043p < 001
. . 20
30
40
Percentage
50 change
60
70
80
90
In L,
Fig. 3. Fluid-filtration rate on the ordinate is plotted against percentage change in cp on the abscissa. The open circles indicate the data points obtained with acid-etched dentine and the sohd circles designate data obtained from unetched dentine.
382
D. H. PASHLEY et al
between PBS after 5 mm versus 1.5 per cent albumin in PBS at 5min). When the experiments were repeated on dentine which had been acid-etched on both sides, the results were similar (Fig. 2 top) to those in the presence of a smear layer, although only the 5-min values were statistically different from control values (p < 0.025). Generally, the reductions in L, were less in the acid-etched groups than the unetched groups although the differences did not reach statistical significance. Re-acid-etching the acid-etched dentine after 5-min air blasts restored the L, back to control values (not shown). The smaller decrease in Lp seen in acid-etched dentine was due to the solubtlizing effect of the higher rates of fluid flow seen in acidetched dentine (Fig. 3). The inverse relationship between dentine filtration rate and the fall in dentine L, (Fig. 3) suggests that the effect of evaporation may have been as great in both unetched and acid-etched dentine but that the putative precipitate was more quickly dissolved in the acid-etched group (i.e. the group that had a high fluid flow). DISCUSSION
By measuring changes in fluid filtration rates across dentine discs devoid of odontoblasts, in response to different evaporation time periods, the effects of evaporation on dentinal fluid were separated from those that produce aspiration of odontoblasts, in uiuo. The results implicate the presence of both inorganic salts and protein as responsible for up to a 50per cent reduction in dentine hydraulic conductance (Fig. 2). When dentine was filled with distilled water, there were no changes in dentine hydraulic conductance (L,) in either unetched or acid-etched dentine in response to air blasts of up to 5 min. Thus, air bubbles or air-locks in the tubules could not be responsible for decreasing dentine permeability. Presumably, under these conditions, the same amount of water was evaporating from these discs as was evaporating from subsequently studied discs; there was no change in L, because of evaporation of water from water does not change its composition. However, when inorganic salt solutions were used (PBS), evaporation of water presumably led to an increase in the concentration of the remaining salts, leading to crystalization or precipitation of unknown salts when their components exceeded their respective solubility products. More evaporation of water would occur at 5 than at 0.5 min, hence the larger reduction in L, after 5 min in both acid-etched and unetched dentine (Fig. 2). Dentinal-fluid protein concentrations are approx. 25 per cent that of plasma (von Kreudenstein, 1955; Haldi, Wynn and Culpepper, 1961; Pashley et al., 1981), and dog-plasma total protein is approx. 6 g per cent; thus, an albumin concentration of 1.5 g per cent simulates the protein concentration of dentinal fluid collected in vivo. In unetched dentine, 1.5 g per cent albumin in water produced reductions in L, at all time periods (p < O.OOS), presumably due to the evaporation of water producing a local increase in albumin concentration to the point where it exceeded its solubility in water and formed a precipitate on or in the smear layer.
Surprisingly, when the same concentration of albumin was dissolved in PBS rather than water, the result was not additive of the two separate effects but was similar to that obtained from either alone. Thus, at each time period there was a progressive decrease in L, (p < 0.005). Although the results of albumin plus PBS were not statistically different from albumin alone, there was a tendency at every time period for the reductions in L, for the albumin plus PBS to be less than those obtained with albumin alone. This may be due to the well-known increase salts produce on protein solubility (Mahler and Cordes, 1971). Thus, the simultaneous rise in salt and protein concentrations may facilitate higher protein solubilities than would be possible in water solutions of albumin. The lower reduction in L, in acid-etched dentine relative to that found in unetched dentine under similar conditions (compare Figs 2 versus 3), is due to a dependence of the decrease in L, on the rate of fluid flow (Fig. 3). Thus. the higher the flow rate, the lower was the decrease in L,. Acid etching removes the smear layer from dentine and permits fluids to flow through the tubules faster than unetched dentine, presumably redissolving both organic and inorganic precipitates. The lower filtration rates obtained in unetched dentine with an intact smear layer would not dissolve the precipitates as readily. Finally, when acid-etched dentine which had shown a large fall in L, was re-etched the L, was restored back to control values (not shown). This suggests that the material occluding dentine, whether organic or inorganic, is acid-labile and is present on or near the dentine surface. Our findings accord with Brannstrbm’s (1960) clinical observations, that prolonged air blasts decrease dentine sensitivity. The hydrodynamic theory of dentine sensitivity (Brlnnstrom et al., 1967) predicts that anything that decreases dentine fluid movement (i.e. dentine Lr) would decrease dentine sensitivity. We have shown that prolonged air blasts do decrease dentine .L,. If odontoblast nuclei were aspirated into the tubules, further reductions in L, would be expected. Acknowledgements-We thank Mrs Shirley Johnston for her secretarial assistance. This work was supported, in part. by NIDR Grant DE 06427. REFERENCES
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