Rate of formation of tertiary dentin in dogs’ teeth in response to lining materials Vladimir Ivanovic, MDS, and Ario Santini, BDS, DDS, Edinburgh, Scotland

DEPARTMENT OF CONSERVATIVE DENTISTRY, UNIVERSITY OF EDINBURGH

The rate of tertiary dentin produced in response to four lining materials placed in standardized cavities in

dogs’ teeth was measured over a period of 119 days. Sterile denatured dentin ash was used as a control

material in an attempt to quantify tertiary dentin formation in response to cavity preparation. In all periods

the amounts of tertiary dentin formed beneath the control material were significantly smaller than those

formed beneath any of the four lining materials. In the first 98 days the rate of formation of tertiary dentin

beneath calcium hydroxide (Calxyl) was significantly greater than that beneath the material containing

corticosteroid (Ledermix cement). In the last 21 days there was no difference in daily rates of tertiary

dentin production beneath all of the lining materials. With all the lining materials there was a highly

significant difference between the daily amounts of tertiary dentin formed in the first 6 weeks and those in

the last 11 weeks.

(ORAL SURC ORAL MED ORAL PATHOL 1989;67:684-8)

T hroughout the functional life of an erupted tooth regular or secondary dentin apposition continues on the pulpal aspect of the primary dentin.’ Tertiary dentin forms as a pulpodentinal response to irritants such as caries, attrition, operative techniques, dental materials, or medicaments2s3 and differs from sec- ondary physiologic dentin in its structure,3-5 its rate of formation,6-8 and its permeability.9* lo

The amount of tertiary dentin formed in response to operative procedures has been shown to be related to cavity depth and more specifically to the quality and permeability29 ” and to the remaining thickness of dentin beneath the cavity.6*‘2

In previous studies tetracyclines have been used in human beings13 and Procion Brilliant Red in mon- keys6* 8 as labeling for tertiary dentin formation. The daily rates of tertiary dentin formation in response to different lining materials as a function of time were not assessed in these studies. A preliminary studyi used tetracycline staining methods to quantify daily rates of tertiary dentin formation beneath lining materials in dogs’ teeth.

The aim of the present investigation was to verify the results of the preliminary study with standard- ized cavity preparations. The daily rates of tertiary dentin produced in response to four commonly used types of lining materials and a control material were measured with a tetracycline labeling technique.

MATERIAL AND METHOD

Three healthy dogs, 3 to 4 years old, with intact dentitions were used. Class V cavities were prepared in all canines, premolars, first and second mandibu- lar molars, and first maxillary molars after anesthe- sia was induced intravenously with ketamine (Ketal- ar, 15 mg/kg body weight) combined with Combelen (0.3 mg/kg body weight). The cavities were prepared in the following manner. A No. 3 round diamond bur was used at 3000 rpm to cut through enamel; then a No. 4 inverted-cone tungsten carbide bur (IS0 No. 010) was used to complete the cavity in dentin. Sterile saline solution was directed onto the head of the bur from a syringe during the cutting procedures. Average tooth measurements were obtained from dry skulls of similar dogs. These measurements were verified in the experimental animals from interdental occlusal radiographs taken immediately preopera- tively. A metal collar was clamped to the shank of the burs so that the remaining dentin thickness beneath the floor of the cavity was not less than 500 cLm.

Experimental group. Four lining materials were used (Table I). After the cavity was prepared the floor of the cavity was washed with sterile saline solution and dried with cotton pellets. The materials were mixed according to the manufacturers’ instruc- tions and were immediately inserted onto the floor of

684

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Rate of formation of tertiary dentin 68s

Table I. Lining materials

Name Description Supplier

Calxyl (batch No. 03254) Sterilized calcium hydroxide in suspension with redistilled Galenika, Zemun, Yugoslavia water and methylcellulose

Cp-Cap Powder: each gram contains calcium hydroxide, 300 mg; zinc Lege Artis, Pharma, Dettenhausen, oxide; 298 mg; Canada balsam, 100 mg; zinconium oxide, 300 German Democratic Republic mg; zinc acetate, 2 mg

Ledermix cement Liquid: eugenol, Peruvian balsam, and Chinoline Powder: each gram contains triamcinolone acetonide, 6.7 mg; Lederle Lab. Div., Hampshire, U.K.

demeclocycline hydrochloride, 20 mg; zinc oxide and calcium hydroxide to 100 mg

ZOE Liquid: eugenol in rectified turpentine oil Zinc oxide, B.P.; eugenol, B.P.

the dried cavity. All cavities in one quadrant were lined with the same material. When the material had set, the cavity was coated with cavity varnish (Copal varnish, DeTreys Division, Dentsply Ltd., Surrey, England), and an amalgam restoration was immedi- ately inserted.

Control group. Sterile dentin ash, mixed with sterile physiologic saline solution immediately before application to the cavity floor, was used as a control material in 12 teeth to estimate the effect of cavity preparation alone on tertiary dentin formation. The third mandibular molars were not prepared. Normal physiologic secondary dentin deposition during the experimental period was measured in these teeth.

To label dentin formation throughout the experi- mental period, 825 mg tetracycline (Reverin; Galen- ika, Zemun, Yugoslavia) was given intravenously in divided doses on 3 consecutive days. The first 3-day regimen was begun immediately after all the cavities had been lined and filled, while the animal was still anesthetized. Subsequent regimens were commenced on the days 29, 50, and 99 postoperatively.

The animals were put to death on day 119 by an intravenous injection of 40 ml of saturated magne- sium sulfate solution. The teeth were cut from the alveolus and stored in 10% buffered formalin. Each tooth was sectioned along the buccooral plane paral- lel to the long axis, through the middle of the cavity, with a diamond rotary cutter at 6000 rpm and copious water spray. Each slice (two per tooth) was then embedded in clear acrylic resin, and sections 40 to 60 pm thick were prepared by grinding. All sections were examined and photographed with a fluorescent microscope (Zeiss; Fluoval, Jenna, Ger- man Democratic Republic) at X63, x160, and ~400 magnification. The mean value of the daily rates of tertiary dentin formation for each lining material was calculated by measuring the distance between

the leading (cavity) edge of consecutive fluorescent lines and dividing by the number of days that had elapsed between the doses of tetracycline. Measure- ments were made at 10 randomly selected sites along the cavity floor and at right angles to the pulpoden- tinal border with a scale in the microscope ocular system. The results were statistically analyzed.

Standardized cavities. The remaining dentin thickness was measured from the cavity floor to the leading (cavity) edge of the first tetracycline line for each specimen. Measurements were made at right angles to the pulpodentinal border at 10 randomly selected sites, and the mean value was calculated for each specimen. To avoid the influence of variation in remaining dentin thickness, only sections with a remaining dentin thickness of 500 to 900 pm were used. This specification reduced the number of specimens from 144 to 120. A further five specimens were damaged in preparation. Therefore 115 speci- mens remained for final analyses.

RESULTS

The mean value of the remaining dentin thickness covering the pulps before tertiary dentin formation was 645 + 166 pm.

Figs. 1 and 2 illustrate tooth sections under ultraviolet light, with tertiary dentin containing tet- racycline-stained lines.

Tertiary dentin deposition was greatest directly beneath the cavity floor and decreased both apically and coronally on either side of the cavity.

The mean daily tertiary dentin deposition in micrometers (x ? SD) for each of the four tested materials and dentin ash during the experimental periods is given in Table II.

The rate of formation and the total tertiary dentin deposited during the experimental periods beneath the control material (dentin ash) were significantly

686 Ivanovic and Santini

Fig. 1. Tooth section under ultraviolet light illustrates tertiary dentin containing tetracycline-stained lines. C, Cavity preparation: Rd, regular dentin; td, tertiary dentin; P, Pulp chamber: a, artifact; LITYOWS indicate first and fourth tetracycline-stained lines. (Original magnification x63.)

Fig. 2. Tooth section under ultraviolet light illustrates decrease in distance between fluorescent lines as well as a thinning of the stained dentin in the tooth section coronal to the cavity. CA, Cavity preparation; rd, regular dentin; p, pulp chamber; arrows indicate first and fourth tetracycline-stained lines. (Original magnification X 160.)

less than beneath any of the lining materials (Stu- dent’s t test, p < 0.001).

There was no significant difference at any mea-

In all four periods tertiary dentin formation was surement period in the rate of formation of tertiary dentin beneath cavities lined with the material con-

greatest beneath cavities lined with calcium hydrox- ide (Calxyl) and least beneath cavities lined with the

taining calcium hydroxide and zinc oxide (Cp-Cap)

material containing corticosteroids (Ledermix and zinc oxide alone (ZOE) (Student’s t test,

cement). p > 0.05).

In the first period (days 0 to 28) and third period

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Rate of formation of tertiary dentin 667

Table II. Daily rate of formation of tertiary dentin in response to lining materials

Postoperative observation period

Lining material No. of samples Days O-28 Days 29-49 Days 50-98

Calxyl 29 4.2 + 0.8* 4.7 f 0.9* 2.6 f 0.4* Cp-Cap 28 2.8 + 1.0 3.1 f 1.1 2.2 f 0.8 ZOE 30 2.7 + 0.6 3.0 ” 0.5 1.9 f 0.3 Ledermix cement 28 1.7 f 0.4 2.0 f 0.4 1.1 k 0.1 Dentin ash 21 1.2 f 0.3 1.4 f 0.3 0.7 * 0.3

Days 99-119

2.6 + 0.6* 2.4 k 0.6 2.1 + 0.4 1.7 zk 0.3

0.8 k 0.4

*Expressed as micrometers per day (mean _t SD).

(days 50 to 98) there was a significant difference only in the amounts of daily formed tertiary dentin beneath the calcium hydroxide material and the material containing corticosteroid (Student’s t test, p < 0.005).

In the second period (days 29 to 49) there was a significant difference between the calcium hydroxide material and the other three materials (Student’s t test, p < 0.01). By 99 days postoperatively there was no significant difference in the amounts of tertiary dentin under cavities lined with calcium hydroxide, zinc oxide alone, and the material containing calci- um hydroxide and zinc oxide (Student’s t test, p > 0.1).

DISCUSSION

In all experimental cases the triple dose of tetracy- cline produced a distinct line within dentin; this line allowed the quantification of tertiary dentin produc- tion during the four experimental periods. In the unprepared teeth these lines were so close together that the small amounts of physiologic secondary dentin produced in each experimental period could not be accurately measured. Compared with the unprepared teeth, control cavities showed an increased amount of tertiary dentin deposited beneath their denatured dentin ash. Cavity prepara- tion under water coolant is known to stimulate odontoblasts to produce reparative dentin,15T I6 and the present findings show that even at 119 days the rate of tertiary dentin formation exceeded physiolog- ic amounts.

The triple tetracycline staining lines and the amounts of tertiary dentin formed between these lines are broadest directly beneath the cavity floor. Coronally and apically the lines and intervening dentin are narrower (Fig. 2). This pattern is inter- preted as indicating the zone of stimulation to be an arc, with the most intense reaction being directly beneath the cavity along the plane of the dentinal tubules. This phenomenon was observed in all speci- mens and is therefore unlikely to be a consequence of the plane of section.

The rate of formation of tertiary dentine in the first period (days 0 to 28) was always less than the rate in the second period (days 29 to 49) with respect to all four materials and dentin ash. This observation is in agreement with previous reports7~8,17 that showed an initial reduction in dentin formation after cavity preparation in dogs’ teeth. A normal or slightly accelerated rate of dentin formation after this initial reduction was considered to be a favorable response.‘* Although all four materials showed an increase in dentin formation in the second as com- pared to the first period, the rates of formation in every case and with every material were much greater than the rate of physiologic secondary dentin produced in the unprepared teeth. Caution must therefore be exercised in interpreting this increased rate of deposition as a return to normal cellular function.

With all four materials there was a decrease in the rates of deposition in postoperative days 50 to 98 as compared to the period of days 0 to 49 (Student’s t test, p < 0.001). This decrease is interpreted as a reduction in intensity of the stimuli both temporary with respect to cavity preparation and spatially, with a barrier effect being produced by tertiary dentin. After 98 days there was significantly less dentin formed beneath the material containing corticoste- roid than beneath the other three lining materials. This difference could be because the corticosteroid suppresses the pulp tissue response. Reports on the effects of corticosteroids placed in deep cavities are conflicting. Odontoblast atrophy and reduction of dentin matrix formation have been reported after corticosteroid application. I9 This inhibition of dentin formation was not confirmed by other research workers.20~21 Corticosteroids are said to inhibit den- tinogenesis** by affecting the function of ‘mesenchy- ma1 cells. However, delay in the recovery of the injured connective tissue occurs only in the first few postoperative days. Once cellular function has been reestablished, corticosteroids are without effect.23 The present study confirms the findings of previous authors24-27 who showed that corticosteroid does not

688 Ivanovic and Santini

completely arrest dentin formation when applied to the base of deep cavities.

Calcium hydroxide is regarded as a special mate- rial’ that allows dentinogenesis to continue without qualitative or quantitative changes. The present study shows that the rate of formation of tertiary dentin was greatest beneath calcium hydroxide and that even after 98 days this rate was equivalent to that of the other three materials in the first period (1 to 21 days). The greater total amount of tertiary dentin formed beneath calcium hydroxide may be undesirable.3 It is perhaps pertinent to state that the mode of action of calcium hydroxide remains unknown. Indeed the cause of irritation from any of the used lining materials remains to be identified. In the case of healthy dentin this cause could be cutting techniques, drying methods, chemistry of the materials and their usage, or microleakage associ- ated with the set material and any accompanying filling.

Many articles on cavity lining materials commend the promotion of reparative dentinogenesis,3, 5, ‘3 25 because tertiary dentin is less permeable and is therefore protective to the p~lp.‘~~ “1 24, 28 Further experiments are being conducted to relate pulp cell dynamics to tertiary dentin production over the experimental period and to assess the permeability of tertiary dentin produced beneath the lining materials used in the present experiment.

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Reprint requesa IO. Dr. A. Santini University of Edinburgh Department of Conservative Dentistry High School Yards Edinburgh EH 1 I NR