A new in vitro technique for studies on tooth permeability

,414~oralBid. Vol. 13,pp. 1057-1065, 1968. Pergamon Press. Printedin Gt. Britain.

A NEW IN VITRO TECHNIQUE FOR STUDIES ON TOOTH PERMEABILITY L. V.

SMITH

and J. P.

DEVINCENZO

School of Dentistry, Loma Linda University, Loma Linda, California, U.S.A. Summary-An in vitro technique for studying tooth permeability, while attempting to maintain the physiologic conditions present in in vivo experiments, was developed. Freshly extracted teeth were perfused with a tissue culture medium and maintained in.an air incubator with the humidity near 100 per cent and a temperature of 37°C. Appropriate test solutions of Naz2 P, and Feb9 were placed in a plastic cap attached to the crown with tape and a special wax.‘A glass T-latex assembly was attached to the apical ) of the tooth and served as a perfusate collecting device.

Every hour during the perfusion period, washings of the coronal 3 of the root were taken to determine the effectiveness of the coronal and root seals. Data from the washing revealed the difficulty in obtaining isotope-impermeable seals and the significant effect which leakage could have on overall results. This technique could be used to study a variety of factors which mighhtinfluence enamel and dentine permeability. INTRODUCTION THE QUESTION

of whether the tooth, a seemingly inert, dense structure could be permeable to certain substances has long intrigued dental investigators. Many have studied factors, such as tooth metabolism or crystal formation, which could influence this permeability. Among the earliest were BUNTING and RICKERT (1918), who noted that fluid movement through teeth could be controlled by osmotic differences. FISH (1933), in the early 1930’s utilized dyes in both in vivo and in vitro experiments with several species of animals including humans. He observed differences in inward and outward flow. He also noted that in comparable experiments the dentine and enamel were penetrated about half as readily under in vitro conditions. Few new insights into the permeability of teeth were gained until the introduction of radioisotope technology. Using freshly extracted teeth, WAINWRIGHT and LEMOINE (1950) demonstrated autoradiographically rapid permeability of tooth structures. SOGNNAES and SHAW (1952) and SOGNNAES, SHAW and BOGOROCH (1955), utilizing several radioisotopes, studied the outward and inward movement in the teeth of dogs and monkeys. The teeth of monkeys were isolated with a rubber dam and a plastic tube was sealed to each tooth crown with sticky wax. Intravenously injected isotopes were detected in these coronal containers, demonstrating an outward flow through the intact tooth. These investigators felt that all of their data could be explained on the basis of simple diffusion. In an attempt to eliminate the necessity of a coronal container for the isotope, BARTELSTONE(1951) dipped the canine teeth of cats into a solution of P; radioactivity in the region of the thyroid gland was noted within 2 hr. 1057

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L. V. S~~ITH AM)J. P. DEV~CENZO

Recent interest has focused on changes in permeability induced by mechanical or physical alterations of the tooth. STOWELLand TAYLOR(1964) found that the penetration of 1131was increased under the influence of a positive electrical potential. KAPUR, FISCHERand MANLY(1961) found an increased penetration of lactate buffer when the enamel surface was roughened, while topical applications of NaF reduced the penetration of the buffer. Due to the high concentrations of radioisotopes required in studies on the permeability of teeth, the possibility is always present that the data obtained and their subsequent interpretation may be influenced, in part, by contamination. FREMLIN and MATHIESON(1961) have illustrated the care that must be taken to prevent radioactive contamination and the possible distortion of results which might follow. It was found that radioactive solutions could migrate over greased surfaces, under such seals as dental wax, silicone grease, pitch, rubber adhesives, varnish, and “araldite” resin electroplated with silver. This migration occurred in a vertical as well as in a horizontal direction. Although several substances appeared to be satisfactory in some cases, they were not satisfactory in all cases. This evidence suggested that it may be extremely difficult to obtain a routinely perfect coronal seal on the enamel. Since a perfect seal has not yet been found, a continual check on the seal would be essential to a correct interpretation of the data. Most in vitro studies on the permeability of teeth have had little regard for maintaining the tooth in a normal physiologic or metabolic state, while in vivo studies have been handicapped by several limitations. Some of these difficulties involve controlling leakage of radioisotopes, checking for contamination, manipulating the internal and external environment of the teeth, and requiring excessive quantity of radioactive material. An in vitro system which could overcome the disadvantages of the in vivo approach, while maintaining normal tooth physiology, would greatly expand the capabilities for studies on tooth permeability. DEVINCENZO(1968) reported that he had developed a perfusion procedure capable of maintaining active pulp tissue metabolism in immediately extracted intact human teeth for periods up to 3 days. This in vitro system has served as a basis for the development of a technique to study tooth permeability. It is the purpose of this report to describe a procedure which, while attempting to maintain the teeth in as near normal in vivo condition as possible, will allow the study of ionic and molecular movements through intact teeth, and, at the same time, monitor continuously the integrity of the coronal seal on the enamel. METHODS AND MATERIALS The basic method for tooth perfusion used in this technique is that described by DEVINCENZO(1968). Two 30 gauge stainless steel needles were passed from the apex to the pulp horns of freshly extracted teeth from young orthodontic patients, age 11-18 (Fig. 1). Appropriate sizes of polyethylene intramedic tubing connected the needles to a glass manifold containing the tissue culture medium. The medium consisted of Waymouth’s MB 752/l (Microbiological Associates, Albany, California) plus 10 per

A NEW

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Vitro

TECHNIQUE

FOR STUDIES

ON TOOTH

1059

PERMEABILITY

cent foetal bovine serum to which was added 100 units penicillin G, 100 pg streptomycin sulphate and 5 pg amphotericin B per ml. The polyethylene tubing from the manifold traversed a small rubber stopper-glass T assembly before it reached the tooth apex. A l-in length of +-in. i.d. latex tubing joined one arm of the glass T to the apical Q of the tooth. Before placing the tubing over the tooth apex, a small groove was made around the root approximately 4 mm

PERFLJSATE EXIT

FIG. 1. An illustration of the permeability apparatus. Samples were collected from perfusate exit and the “washing area”. The total volumes of the isotope container and the glass T-latex tubing assembly were O-10 and O-30 ml respectively. Numerals 1,2, 3, and 4 represent possible paths which an isotope could follow.

from the tooth apex. A stainless steel O-012 orthodontic ligature wire was then placed around the latex tubing and twisted tightly, compressing the tubing into the groove. Thus the first arm of the glass T was connected to the apex of the tooth, the second arm was sealed with a rubber stopper, containing the tubing, while the third arm of the T served as the perfusate exit. A container for the radioisotope was made from a commercially available plastic cone (Kerr Manufacturing Co., Detroit, Michigan), and adapted to the crown of the tooth with masking tape and sealed with a wax consisting of 1 part dental sticky wax and 1 part Picein-80 (Hamburger Gummi-Waaren, N.Y.). To obtain the best possible seal a thin layer of this wax combination was first applied to the surface of the tooth between the cemento-enamel junction and the height of contour. The masking tape was then placed around the tooth. The seal area and the entire isotope container were finally covered with a second layer of wax. A small stream of air was delivered continuously to the area of wax application in an attempt to minimize heat damage to the tooth. The entire tooth assembly, which was fabricated within 15 min of tooth extraction, was housed in an air incubator with the humidity near 100 per cent and a temperature of 37°C. A gas mixture of 9.5 per cent 0, and 5 per cent CO, was continuously

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L. V. Sm

ANDJ. P. DEVINCENZO

delivered into the incubator. A flask containing the tissue culture medium was located outside the incubator at a distance above the tooth sufficient to produce pressure equivalent to 20 mm mercury. The rate of perfusion was about 4 ml/tooth per hr. As the perfusate exited from the glass T it was collected; the collecting vial was changed every hour. The area between the coronal seal and the root seal was washed every hour with approximately 4 ml of Waymouth’s MB 752/l tissue culture medium. The washing and perfusate were collected separately and refrigerated until radioactive determinations could be made. All samples were counted to a preset count of either 1000 or 4000 by deep-well scintillation detection. The isotopes used were Na2*, P31, and Fe68 as ferric chloride, all with approximate activity of 0.5 me/ml. The isotope solutions of Naza and P31were made up to 0.11 M with NaCl. Approximately 0.1 ml of the radioactive solution was required to fill each coronal radioisotope container. The isotopes were delivered to the coronal containers through a 30 gauge needle attached to a mechanical micropipette. Extreme care was taken to avoid contaminating the outside of the coronal container during the tiling process. Particular attention was also given to the expulsion of air bubbles which became entrapped easily in parts of the isotope containers. A visual check on gross leakage was made by observing any change in the level of the meniscus, formed by the isotope solutions, during the experiment. RESULTS

The periodic monitoring of the coronal seal and the possible influence of seal leakage on the interpretation of overall results can best be demonstrated by data obtained on individual teeth. In tooth A (Fig. 2) radioactivity fust appeared in the perfusate after 5 hr of perfusion and gradually increased until the termination of the experiment at 24 hr. Radioactivity appeared in the washing after 12 hr of perfusion and remained at a low, constant level throughout the remainder of the experiment. In tooth B (Fig. 2) radioactivity appeared in the perfusate and washing after 3 hr of perfusion and increased rapidly until the ninth hour of perfusion, The perfusate and washing radioactivity remained at a rather constant level after the ninth hour. In tooth C (Fig. 2) radioactivity appeared in the washing after 5 hr of perfusion and increased throughout the experiment, while perfusate activity failed to appear until the eleventh hour of perfusion. Radioactivity appeared in the washing of tooth D (Fig. 2) after 2 hr of perfusion and increased rapidly, reaching a maximum after 7 hr of perfusion. The perfusate radioactivity produced a pattern similar to that of the washing but followed it by about 5 hr. The permeability of teeth varied with the isotope used. In a small sample of 4 teeth using Fe60, radioactivity failed to appear in the perfusate of any of the teeth during a 24hr period. After 12 hr radioactivity appeared in the washing of one tooth but not in the perfusate. In two teeth using P31 radioactivity failed to appear in the washing or perfusate during a 24-hr period. Tooth B represents the only tooth using Psl in which radioactivity appeared in the washing and perfusate at the same time. In all other teeth, P” appeared in the perfusate before it appeared in the washing.

A NEW in Vitro TBCHNIQIJEFOR STUDIES ON TOOTH PERMEABILITY

TOOTH

A

TOOTH

C

TOOTH

B

TOOTH

D

FIG. 2. Data obtained from individual teeth. P was used with teeth A and B; Naaa was used with teeth C and D. Squares represent the washing, circles the perfusate. Samples with radioactivity less than 10 per cent above background were not plotted. The 10 per cent figure represents radioactivity 10 per cent above background. The ordinate represents the reciprocal of the time in minutes required to reach the present count.

TABLE 1. TIME OF FIRST APPEARANCE OF ISOTOPE (IN HOURS)

Naaa (6 teeth)

I’S’ (6 teeth)

Perfisate Mean Range

5.5 l-11

5.8 3-8

Washing Mean Range

2.5 l-5

7.6 3-12

In four additional teeth, two with sodium and two with iodide, radioactivity was absent in both perfusate and washing.

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L. V. %rrn ANDJ. P. DEVINCENZO

In all experiments using Naza, where radioactivity washing before it appeared in the perfusate. Note the of sodium in the perfusate and washing as compared two teeth where Naz2 failed to appear in the washing perfusate.

appeared, it appeared in the mean time of first appearance with iodide (Table 1). In the it also failed to appear in the

DISCUSSION It has been postulated that the permeability of teeth may be influenced by characteristics of the penetrating ions or molecules such as molecular size, electrical charge, and spatial configuration. Certain chemical, physical, and electrical changes (STOWELLand TAYLOR,1964; KAPUR, FISCHERand MANLY,1961) of teeth have been found to alter permeability. FISH (1933), SOGNNAES and SHAW(1952), TARBET(1964) and TARBETand FOSDICK(1964) have noted differences between in vivo and in vitro studies on tooth permeability. Greater and more rapid penetration was noted in in vivo experiments. If in vivo experiments are not workable because of inherent limitations, it would seem desirable to maintain the in vitro counterpart as near physiologic conditions as possible. Of all in vitro studies on tooth permeability FISH (1933) probably maintained the tooth as near physiologic conditions as anyone since that time. He used freshly extracted teeth which were kept at 37°C in a humid atmosphere. The technique described herein, not only maintains freshly extracted teeth at 37°C in a 100 per cent humid atmosphere but, in addition, utilizes a perfusion technique which supplies the pulp with nutrients capable of maintaining high adenosine triphosphate levels for extended periods of time. In most studies on tooth permeability utilizing radiosotopes an isotope container has been sealed to the enamel of the tooth. The most commonly used sealant has been a dental wax. ATKINSON(1947) attempted to duplicate and evaluate such previously used sealants as waxes, dental cements, adhesives, and those of a pressure type and found that they all eventually leaked. He concluded, after noting that earlier investigators had failed to test the adequacy of their seals, that the data of all these previous experiments could have been distorted. He then developed a seal consisting of a rubber tube which was tied to the root of the tooth with either annealed wire or silk floss. Each seal was checked for leakage at the beginning of the experiment and was considered adequate if an initial check of the electrical resistance across the seal was infinity when tested at 200 V a.c. and d.c., and if no leaks could be detected when the tubes were subjected to water and air pressure of 10 psi. However, since seals were not checked during or at the end of the experiment, their adequacy was assured only at the beginning. Any breakdown in the seal during the experiment would not have been detected. In the present investigation dental sticky wax was found to be unsuitable for sealing the isotope container to the tooth because it did not adhere well to the tooth surface in a 100 per cent humid atmosphere, even though the tooth was dried prior to sealing. Other types of more adherent waxes, silicones and plastics were found to require either higher melting temperatures or longer setting times than seemed

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desirable. It was found that a combination of dental sticky wax and Picein-80 exhibited the best adherent quality and melting temperature, and it was therefore used in this procedure. There are a number of possible pathways, using this technique, by which a radioisotope could appear in the washing area. Three paths are illustrated in Fig. 1. It is possible that the seal could have been inadequate at the time of the placement. In this case one would expect to observe increasing radioactivity in the washing soon after the isotope containers were filled. Tooth D might be an example of this leakage (path 2). It is also possible that the “bond” between the enamel and the wax could deteriorate after a period of time producing a delayed onset in the radioactivity of the washing with an increasing amount of activity thereafter. Perhaps this occurred in tooth C. That this could in fact occur is supported by the work of BERGMAN (1963) and BERGMANand SILJESTRAND (1963), who noted small droplets emanating from the enamel surface of freshly extracted teeth. These water droplets might accumulate in the enamel-wax interface and eventually loosen the seal. They also reported that an outward flow could be measured. This suggests that it may be impossible to seal, with conventional methods, the crowns of freshly extracted teeth for long periods of time. The radioisotope could actually penetrate the coronal enamel and dentine (path I), and, after reaching the pulp chamber, it could either traverse the dentinal tubules in the root area (path 3) or seep through the root seal (path 4) to reach the washing area. This probably occurred in tooth A. There can be no question that a given radioisotope has penetrated a tooth if radioactivity appears in the perfusate before it appears in the washing. It would be physically impossible for an isotope which has leaked through the coronal seal, to reach the perfusate without first being detected in the washing. By comparing the time of first appearance and the intensity of radioactivity in the washing and the perfusate, it should be possible to determine for each tooth if the radioisotope has taken path 1 or 2 (Fig. 1). The data obtained from the washings demonstrate the importance of monitoring periodically for radioactive contamination. The data also indicate the ease with which radioisotopes can pass through supposedly good mechanical seals and significantly influence the interpretation of overall results. For example, had washings not been taken, teeth C and D would have given erroneous appearance of being permeable to the isotope. FREMLINand MATHIESON(1961) have also had difficulty in containing isotopes within a circumscribed area. They investigated sealing properties of a number of substances. In spite of the precautions which they took, radioisotopes moved with ease through a variety of mechanical barriers as well as in a vertical direction. Their findings do not support the observation of BARTELSTONE (1951) that radioisotopes do not travel vertically. Our data are in agreement with FREMLINand MATHIESONon the ease with which radioactive leakage can occur. The marked influence that leakage can have on the interpretation of results suggests that perhaps previous conclusions using radioisotopes to study movements through teeth, should be re-evaluated in the light of possible contamination.

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L. V. SMITHANDJ. P. DMNC~NZO

The data obtained using this technique indicate that 11~1 will penetrate some human teeth in a 5-hr period. It is possible that the perfusate radioactivity in tooth B resulted from leakage of the seal since radioactivity appeared in both the perfusate and washing after 3 hr of perfusion. The finding that radioactive iodide did not penetrate some teeth within a 24hr period concurs with that of STOWELL and TAYLOR (1964). Under the conditions of this experiment, FeJB as ferric chloride does not appear to penetrate the tooth in 24 hr. This might be expected since ferric salts form precipitates with such anions as carbonate and phosphate. Although the results obtained with NaZ2 can be explained entirely on the basis of leakage, the possibility that Na** penetrated the tooth cannot be eliminated. Since some of the first experiments were those using NaZ%,inexperience in the sealing technique would probably account for some sodium leakages. However, distinct permeability patterns clearly emerge for the three isotopes used in this study despite frequent leakage of the coronal container. A direct comparison between the amount of radioactivity in the washing and in the perfusate would be useful, but only if it could be shown that the collected radioactivity was directly proportional to the total activity for each region. This relationship has not been established. The use of this technique would permit the rigid control of the internal and external environment of a tooth undergoing permeability studies. For example, the role of a metabolizing pulp on tooth permeability could be investigated by using a specific antimetabolite in the perfusing medium. Possible changes in tooth permeability resulting from alterations in the external environment of the enamel, by such substances as acids or micro-organisms, could be detected. By removal of a portion of enamel, factors which might influence the permeability of dentine could be investigated. Acknowledgements-This investigation was supported by U.S.P.H.S. Grants DE-02169 and 5 SO1 FR 5302 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland. R&ann&Une methode in vitro a et6 mise au point pour etudier la permQbilit6 dentaire, tout en maintenant des conditions physiologiques. Des dents fralchement extraites sont perfus& avec un milieu utilid en culture de tissu et maintenues dans un incubateur A une humiditt de p&s de 100 pour cent et une temperature de 37°C. Des solutionstests de Na”, Ir3* et Fe59 sont pla&s darts une capsule en plastique attach&s a la couronne avec de l’adh&sif et une tire spt?ciale. Un dispositif en verre est !ixC au l/3 apical de la dent et permet de recueillir les solutions. Des prelevements liquides des 213 radiculaires sont recueillis toutes les heures, pendant la p&iode de perfusion, pour verifier l’efficacitd des joints radiculaire et coronaire. Les resultats demontrent la difficult6 de r6alisation d’un joint impermeable aux isotopes et l’importance de la permeabilite sur l’ensemble des resultats. Cette technique peut etre utilis6e pour l’btude de divers factcurs pouvant intervenir dans la permeabilite de l’tmail et la dentine. Zwnmmenfassung-Es wurde eine in aitro-Technik zur Priifung der Zahnpermeabilitlt entwickelt, welche darauf abzielt, die bei in Go-Experimenten vorhandenen physiologischen Redingungen aufrecht zu erhalten. Frisch extrahierte Zlhne wurden mit einem

A NEWin Vitro TECHNIQUE

FORSTUDIESON TOOTHPERMEABILITY

106.5

Gewebekulturmedium perfundiert undin einem Luft-Inkubator mit einem Feuchtigkeitsgehalt von nahezu 100 Prozent und einer Temperatur von 37°C aufbewahrt. Geeignete Testliisungen von NazZ. 113’ und Fe59 wurden in eine Plastikkanoe gefilllt. die mit Klebestreeifen und Spezialwachs an der Krone befestigt wurden. Am*ipikalen drittel des Zahnes wurde eine Glasvorrichtung angebracht, welche es erlaubt, die perfundierende Fltissigkeit zu sammeln. WIhrend des Perfusionsversuches wurden die koronalen zwei Drittel der Wurzel stiindlich abgespiilt, urn die Wirksamkeit der Abdichtungen an der Krone und der Wurzel zu iiberpriifen. Die Untersuchung der Waschfliissigkeiten deutet auf die Schwierigkeit hin, einen isotopendichten AbschluR zu erreichen; in gleicher Weise zeigt sich der erhebliche EinfluB einer Undichtigkeit auf die Ergebnisse. * Diese Versuchstechnik kormte zur Untersuchung verschiedener Faktoren benutzt werden, welche die Schmelz- und Dentinpermeabilitlt zu beeinflussen vermiigen.

REFERENCES ATKINSON, H. F. 1947. An investigation into the permeability of human enamel using osmotic methods. Br. dent. J. 83, 205-214. BARTELSTONE, H. J. 1951. Radioiodine penetration through intact enamel with uptake by bloodstream and thyroid gland. J. dent. Res. 30, 728-733. BERGMAN,G. 1963. Microscopic demonstration of liquid flow through human dental enamel. Archs oral Biol. 8, 233-34.

BERGMAN,G. and SIWESTRAND,B. 1963. Water evaporation

in vitro from human

dental enamel.

Archs oral Biol. 8, 37-38.

BUNTING,R. W. and RICKERT, U. G. 1918. The tooth, a permeable membrane. Nat. dent. A. J. 5, 519-526. DEVINCENZO,J. P. 1968. An organ culture technique for maintaining the pulp tissue of intact human teeth. Expl. CeN Res. In press. FISH, E. W. 1933. An Experimental Investigation of Enamel, Dentine, and the Dental Pulp. John Bale and Sons and Danielsson, London. FREMLIN,J. H. and MATHIESON,J. 1961. A microchromatographic study of the penetration of enamel of CXlabelled glucose. Archs oral Biol. 4, 92-96. KAPUR, K. K., FISCHER,E. and MANLY, R. S. 1961. Effect of surface alteration on the permeability of enamel to lactate buffer. J. dent. Res. 40, 1174-l 182. SOGNNAES,R. F. and SHAW, J. H. 1952. Salivary and pulpal contributions to the radiophosphorus uptake in enamel and dentin. J. Am. dent. Ass. 44,489-505. SOGNNAES, R. F., SHAW, J. H. and BOGOROCH,Rita. 1955. Radiotracer studies on bone, cementum, dentin and enamel of rhesus monkeys. Am. J. Physiol. 180,408420. STOWELL,E. C. and TAYLOR,J. B. 1964. Influence of electrical potential on ion migration in teeth. 2. Quantitative measurements of I-131 penetration by an acid-leaching technique. J. dent. Res. 43, 175-186.

TARBET,W. J. 1964. A study of the permeability and posteruptive maturation of human enamel. Ph. D. Thesis, Northwestern Univ. TARBET,W. J. and FOSDICK,L. S. 1964. In vivo permeability of teeth. J. dent. Res. 43,872. Abs. 303. WAINWRIGHT,W. W. and LEMOINE,F. A. 1950. Rapid diffuse penetration of intact enamel and dentin by carbon14-labeled urea. J. Am. dent. Ass. 41, 135-145.

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