Archives of Oral Biology 45 (2000) 931–935

The permeability of human dentine in vitro and in vivo

N. Vongsavan 1, R.W. Matthews 2, B. Matthews *Department of Physiology, School of Medical Sciences, Uni6ersity of Bristol, Uni6ersity Walk, Bristol BS8 1TD, UK

Accepted 6 June 2000

Abstract

Experiments in cats have shown that Evans blue dye diffuses at a greater rate into dentine in recently extractedteeth than in vivo. These experiments have now been repeated in man and similar results were obtained except that,after applications in vivo, visible concentrations of the dye were present in the dentine, and in a few cases, even inthe pulp. It is concluded that, as in the cat, the diffusion in vivo was impaired by outward flow of fluid in the dentinaltubules but the mean velocity of flow in the human dentine was less than that in the cat. © 2000 Elsevier Science Ltd.All rights reserved.

Keywords: Dentine permeability; Dentinal fluid; Evans blue

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1. Introduction

Both human (Anderson and Ronning, 1962) and cat(Vongsavan and Matthews, 1991) dentine were freelypermeable to Evans blue dye when tested in recentlyextracted teeth. However, in the cat, the rate of diffu-sion of the dye through dentine was much lower whenit was applied in vivo (Vongsavan and Matthews, 1991)and this difference was attributed to the presence of anoutward flow of fluid through the exposed dentinaltubules in vivo which opposed inward diffusion(Vongsavan and Matthews, 1992a). The present experi-ments sought to compare the rates of diffusion ofEvans blue through human dentine in vitro and in vivo.

2. Materials and methods

The experiments were carried out on 12 first orsecond premolar teeth of six patients (age, 12–14 years)who were to have the teeth extracted under local anaes-thetic as part of their orthodontic treatment. Patientswere selected who were to have either both upper orboth lower first premolars extracted. The tooth on oneside was tested in vivo and the contralateral tooth invitro. The experiments, which were carried out in theDivision of Oral Medicine of the Bristol Dental Hospi-tal, were approved by the Hospital Ethical Committee.Informed consent was obtained from each patient andtheir parents or guardians.

At the appointment, at which the teeth were to beextracted, the tooth to be tested in vivo was anaes-thetized with local anaesthetic. The anaesthetic (mepi-vacaine hydrochloride, 0.12 mol/l; Scandonest, 3%;Septodont, Dorking, UK) contained no vasoconstric-tor. For an upper tooth, approx. 1 ml was infiltratedbuccally and 0.5 ml palatally over the roots. For alower tooth, 1–2 ml was injected to block the ipsilateralinferior alveolar nerve. Dentine was then exposed, asdescribed below, at the tip of either just the buccal cuspor both buccal and palatal cusps of the tooth. An

* Corresponding author. Tel.: +44-117-9287816; fax: +44-117-9288923.

E-mail address: b.matthews@bris.ac.uk (B. Matthews).1 Faculty of Dentistry, Mahidol University, Yothi Street,

Bangkok 10400, Thailand.2 Department of Oral and Dental Science, University of

Bristol, Lower Maudlin Street, Bristol BS1 2LY, UK.

0003-9969/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0003 -9969 (00 )00079 -0

N. Vongsa6an et al. / Archi6es of Oral Biology 45 (2000) 931–935932

acrylic cap with a hole (2-mm diameter) over the tip ofeach cusp was then cemented over the tooth crown withcalcium hydroxide (Life; Kerr Ltd., Peterborough,UK). The cap had been constructed before the appoint-ment on a plaster model of the patient’s dentition. Thespace under the cap left by the removed enamel anddentine was filled with a drop of Evans blue (52 mmol/l) solution, which was made up in sterile isotonic saline.The solution was passed through a 0.5-mm-pore filterimmediately before use. After 15 min, the Evans bluewas washed out and the tooth was extracted withforceps. The contralateral tooth was then also extractedunder local anaesthetic. This tooth was prepared andexposed to Evans blue in the same way within 1 hr ofthe extraction.

An axial, buccopalatal, longitudinal section, approx.0.5-mm thick, was cut through the each cusp with adiamond disc under Ringer’s. The sections were dehy-drated overnight in 100% ethyl alcohol, cleared inmethyl salicylate and examined under a microscope(Wild, type M400) with variable darkground illumina-tion. The maximum distance the dye penetrated intothe dentine was measured.

Dentine was exposed in one of two ways. In somecases, 2–3 mm of enamel and dentine were removedfrom the tip of a cusp with a diamond bur in anair-rotor handpiece under a constant stream of water.The exposed dentine surface was then etched withorthophosphoric acid (4.6 mol/l) for 30 s to remove thesmear layer. In others cases, the dentine was exposed byfracturing. A groove was cut in the enamel 2–3 mmbelow the cusp tip with a diamond bur under a streamof water, then the cusp tip was fractured off with finesurgical shears under a drop of saline. In each pair ofteeth from the same patient, the same procedures wereused to expose the dentine for testing in vivo and invitro.

3. Results

In all of the teeth tested in vitro, Evans blue pene-trated the exposed dentine, and in all but one it wasvisible in the underlying pulp horn (Fig. 1A). Much lessdye was visible in the teeth that were tested in vivo,under two of the cusps there was none in either thedentine or the pulp apart from a very thin layer ofstained, decalcified matrix at the etched dentine surface;in the other five, just a few of the tubules under thecenter of the area of exposed dentine were stained (Fig.1B) and in two of these, there was faint staining of thepulp horn. The minimal penetration of the dye in the invivo experiments contrasted markedly with the muchheavier staining in the teeth that were tested in vitro.

The results of the measurements of the maximumpenetration distance of the dye, together with the thick-

ness of remaining dentine, are given in Table 1. Therewas no significant difference (P=0.1 Student’s t-test)between the mean dye penetration in vitro (2.9 mm;S.D., 0.5) and in vivo (1.9 mm; S.D., 1.4) when mea-sured in this way. The method of exposing the dentinehad no obvious effect on the results.

4. Discussion

The results obtained in vitro are similar to those ofAnderson and Ronning (1962), bearing in mind that inthe present experiments, the dye was applied for only15 instead of 30 min. Anderson and Ronning appliedEvans blue to fractured dentine, but in other prelimi-nary experiments, we have confirmed that similar re-sults are obtained with 30-min applications of the dyein recently extracted, human premolars independent ofwhether the dentine is exposed by fracturing or bydrilling followed by etching with acid. The same wasfound in published work with cat dentine (Vongsavanand Matthews, 1991).

The observation that less dye diffused into humandentine in vivo than in vitro corresponds with theresults of similar experiments in cats (Vongsavan andMatthews, 1991) and it is likely that, as in the cat, thediffusion in vivo was impaired by outward flow of fluidin the dentinal tubules (Vongsavan and Matthews,1992a). However, unlike the cat experiments, dye couldbe detected in the dentine in five of the seven humanpreparations, and in the pulp in two. Furthermore,there was no significant difference between the averagemaximum depth of penetration of the dye when it wasapplied in vivo instead of in vitro. It seems as if a smallproportion of the tubules behaved in vivo in the sameway as the majority did in vitro. These differencesbetween the human and cat experiments in the penetra-tion of the dye in vivo suggest that the mean velocity offlow through the tubules in the human dentine in vivowas less than that in the cat, and that in some of thehuman tubules there was little flow at all.

A lower mean flow velocity in the tubules of humandentine compared with those of the cat could have beendue to the resistance to flow through each tubule beinggreater, the hydraulic conductance of the odontoblastlayer being lower, or the net filtration pressure betweenthe pulp and dentine being lower in the human teeth.As the dentinal tubules tend to be larger in diameter inhuman than cat dentine (Forssell-Ahlberg et al., 1975;Garberoglio and Brannstrom, 1976; Vongsavan andMatthews, 1992a), it seems unlikely that the resistanceto flow through the tubules was greater. Nothing isknown of the hydraulic conductance of the odontoblastlayer in either species under the conditions of theseexperiments. Species differences in either the pulpaltissue fluid pressure or the opposing osmotic effect of

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constituents (e.g. protein) of the pulpal tissue fluidcould have resulted in the net filtration pressure beinglower in the human than that in the cat teeth, althoughthe estimates that have been made indicate that they aresimilar, 1.5 kPa in the cat (Vongsavan and Matthews,

1992a) and 1.4 kPa in man (Ciucchi et al., 1995).Pulpal tissue fluid pressure has been estimated with

the micropuncture technique in the cat and found to beof the order of 0.72 kPa (Tønder and Kvinnsland,1983), whereas in man values in the range 1.4–3.3 kPa

Fig. 1. Photomicrographs of ground longitudinal sections through the crowns of two human premolar teeth. In each case, a solutionof Evans blue had been applied for 15 min to dentine that had been exposed with a bur under the tip of a cusp then etched withacid. The sections were dehydrated and cleared in methyl salicylate. Original magnification, ×42. (A) Recently extracted tooth. TheEvans blue penetrated through the exposed dentine to the pulp. No dye was visible in the pulp. (B) The dye was applied in vivo.The arrow indicates a small area in which a few tubules contained Evans blue. No dye was visible in other parts of the section. Thissection passed through the edge of the pulp and some dentine is superimposed on the outline of the pulp chamber.

N. Vongsa6an et al. / Archi6es of Oral Biology 45 (2000) 931–935934

Fig. 1. (Continued)

have been recorded with a closed cannulation technique(Beveridge and Brown, 1965; Andrews et al., 1972).Pulpal tissue fluid pressure may have been reduced inthe present experiments as a result of pulpal vasocon-striction (Vongsavan and Matthews, 1992b) caused ei-ther by a high level of sympathetic vasoconstrictor tonein the stressed participants or by the mepivacaine. Incats, sympathetic stimulation can cause a reversal of thenormal outward flow of dentinal fluid (Matthews andVongsavan, 1994), which would facilitate inward diffu-sion of the dye. In other experiments in man, cavity

preparation under mepivacaine local anesthesia ap-peared to produce a fall in pulpal blood flow (S.Soo-Ampon et al., unpublished). Pulpal tissue fluidpressure may also have been affected by other factors(Matthews and Andrew, 1995).

Although the method of application of the Evansblue was essentially the same in both the human andthe cat experiments, except that it was applied for 15instead of 30 min in the present experiments, the meth-ods used to expose the dentine were not identical, theheight of the cusp was gradually reduced with a bur in

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Table 1Remaining dentine thickness (RDT) and maximum penetration distance of Evans blue through exposed dentine in human teethtested in vitro and in vivo

Case number In vitroExposed by In vitro

RDT (mm) Penetration (mm) RDT (mm) Penetration (mm)

1 Fracture 2.8 2.8 3.3 02 3.5Fracture 2.5 3.1 0

3.5 3.5Fracture 3.43 3.1Disc and etch4 3.5 3.5 3.6 3.2

2.9 2.95 3.0Disc and etch 3.03.0 3.0Disc and etch 3.06 3.0

Disc and etch7 2.0 2.0 2.3 1.2Mean 3.0 2.9 3.1 1.9

0.5 0.5 0.4 1.4S.D.

the human experiments whereas a disc was used to sliceoff the tip of the cusp in the cats. These techniquescould have disrupted the odontoblasts by differentamounts. Also, it was not possible to fracture off thetips of the cusps of the human teeth as accurately as inthe cats, particularly in vivo.

Thus, no definite conclusion can be drawn about thecause of the higher rate of diffusion of Evans blue intohuman than cat dentine in vivo. Further experimentsare required to define more precisely the properties andlocation of the diffusional barriers in vivo and to obtainquantitative data on rates of diffusion. The results ofthe present experiments emphasize the need for care inpredicting the effects of fluid flow through dentine inman from data obtained from experimental animals(see also Vongsavan et al., 2000; this issue).

Acknowledgements

This work was supported by The Wellcome Trust.

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