touay Jaber, DDS, MSc,” Christiane Mas&v, DrCD, PhD,” and William B. Donohue, DDS, MSc, FRCD(C),’ Montreal, Quebec, Canada UhIVERSITY

OF MONTREAL

AND ST. MARY’S

HOSPITAL

CENTER

The purpose of thus study was to evaluate the action of hydroxyapatite (HA) (Osteogen HA Resorb, GBD Marketing Group Inc., Valley Stream, N.Y.) on the dental pulp of rats. Four upper molar pulps in 45 rats were exposed and capped with synthetic HA (Osteogen) with a stereoscopic microscope. Pulps capped with calcium hydroxide (Dycal, L.D. Caulk Co., Milford, Del.) served as controls. The cavities were filled with amalgam, and the molars on each side of the maxilla were protected by the placement of a pedodontic steel crown. Pulp inflammation and dentin repair were compared by histologic observations and computer image analysis after 7, 14, and 28 days. After 7 days a partial acute pulpitis was observed in specimens treated with Osteogen or Dycal. Reparative dentin formation along the pulp walls was also seen. After 14 days the pulpitis was more extensive in the Osteogen-treated teeth than in the control teeth. Dentin formation as measured by morphometric analysis was more pronounced in Osteogen-treated teeth. Neo-odontoblasts were observed after the use of both materials. After 28 days an acute inflammatory reaction was still evident in the Osteogen-treated group. A complete dentinal bridge was observed more frequently with Dycal than with Osteogen. Despite the putative abilities of HA to be osteoconductive, osteogenic, and dentinogenic, the results of this study indicate that it should not be used as a pulp-capping agent because of its tendency to cause scattered dystrophic calcification in the dental pulp, which could interfere with future endodontic treatment. (QRAL SURC ORAL MED ORAL PATHOL 1992;73:92-8)

D

irect pulp capping is often used to preserve pulp vitality when pulp exposure occurs. Several materials have been tested to observe their effect on the dental pulp. Calcium hydroxide is one of the materials frequently used. lp2 Its effects have been studied extensively in rodents and monkeys, and it is one of the most successful direct pulp-capping agents. However, experiments with Ca(OH)z in rats have resulted in conflicting observations. Rowe,’ for example, observed severe pulpal inflammation and the irregular dentin repair of a poor quality. In another study in which Dycal, the commercial brand of Ca(OH)z, was used, dentin formation was dense.2 aDepartment of Stomatology, Faculty ol” Dental Medicine, University of Montreal. bProfessor, Department of Stomatoiogy, Faculty of Dental Medicine, University of Montreal. CProfessor, Department of Stomatology, Faculty of Dental Medicine, University of Montreal, and Division of Oral and Maxillofacial Surgery, St. Mary’s Hospital Center. 7/E/32139

Nevertheless, many clinicians consider Ca(OH)z to be the best available material for pulp capping in hu. man beings. Although numerous materials have been used for direct pulp capping in rats, no studies with hydroxyapatite (HA) as a pulp-capping agent in rats have yet been published. HA is often used in edentulous patients with mandibular atrophy for preprosthetic ridge reconstruction. New bone formation has been observed around HA granules in human beings3 and mice4 after the material is first soaked in bovine skin collagen and then implanted into muscle. HA is considered to have an osteoconductive. and possibly under certain circumstances an osteoinductive potential. Because of its osteoconductive quality, it could be considered to be a good potential pulp-capping agent. However, few studies have been published with HA as a pulp-capping agent. 5,6 These two studies resulted in controversial results in that, although the same brand of HA was used in monkeys in both reports, only one of the

93

Dental pulp reaction to hydroxyapatite

Volume 73 Number 1

Table I. Degree of pulp inflammation

Table II. Dentin bridge formation

after pulp

at exposure site

capping

Osteogen Dycal Osteogen Dycal Osteogen Dycal

Material

No. of teeth

Material 7 7 14 14 28 28

12 18 13 16 13 12

0 1 0 2 0 5

12 16 11 14 0 0

0 0 0 0 8 6

0 1 2 0 5 1

studies reported that HA was a.n acceptable pulpcapping agent.6 We report the reaction of the dental pulp to HA when used as a direct. pulp-capping agent in rats. For the bioanalysis of pulp-capping agents the rat has certain advantages: it is readily available and is easy to handle compared with monkeys. HA is available in three forms7: porous, nonporous, and resorbable. It is also available as free-flowing particles or as a solid block. The granular free-flowing resorbable form (Osteogen HA resorb, GBD Marketing Group Inc., Valley Stream, N.Y.) was selected because of the smaller particles, which allow for better adaption to the dental pulp. MATERIAL

AND METHODS

To study the dynamics of dental pulp healing after direct pulp capping, 4.5 male Sprague-Dawley rats weighing between 200 and 250 gm were separated into three groups of 15 animals each. The animals were housed one per cage and were given ground food (Purina. Chow, St. Louis, MO.) and water at will. Rats were weighed twice per week. This investigation was carried out on 84 teeth to study the dynamics of tissue repair. The animals were killed 7, 14, and 2X days after the experimental procedure. The number of teeth for these sacrifice periods was 12, 13, and 13 teeth in the Osteogen-treated sides and 18, 16, and 12 teeth with Dycal. The time periods were selected because the pertinent literature has reported that a dentinal bridge may be expected after 28 days.2 The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (M.T.C. Pharmaceuticals) (50 mg/kg body weight) and were attached to a surgical table, where the mouth was forced open with special lip and cheek retractors. The two first upper left and right molars were treated with the use: of a stereoscopic microscope (Bausch & Lomb, Rochester, N.Y.), at a magnification of 0.7~ to 3x, i.n the following way. The teeth were cleaned

Osteogen Dycal Osteogen Dycal

14 14 28 28

13 16 13 12

0 0 0 0

12 13 8 5

1 3 5 I

*Incomplete tcomp1ete

with a cotton pellet soaked in a 70% alcohol solution. A cavity was prepared in each tooth with a No. 33% inverted cone tungsten bur with a speed-reduction handpiece under continuous irrigation with sterile 0.8% saline solution and simultaneous suctioning. After the cavities were carefully prepared, the last thin layer of dentin was gently removed with a sharp probe. The exposed pulp appeared as a red dot. The cavities were irrigated with the sterile saline solution and dried with the blunt end of sterile endodontic paper points. Pulps in the experimental side were capped with the Osteogen brand of HA. The pulps in the other side were capped with approximately 0.6 mm in thickness Dycal, and served as controls. The upper third molars were left untreated in order to serve as absolute controls. The cavities were sealed with a thin layer of amalgam (Dispersalloy, (Johnson and Johns,on, East Winsor, N.J.)) (about 1 mm thick) with the use of a miniature amalgam carrier.* Amalgam was selected in preference to other materials, to diminish the probability of a chemical reaction between the two materials. Because retention in the cavities was believed insufficient to retain the amalgam for the required period of time, a No. 6 steel pedodontic steel crown was fitted and cemented over the three molar teeth in each quadrant with zinc phosphate cement. The animals were killed under general anesthesia with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) after 7, 14, and 28 days by intracardiac perfusion with a 10% neutral formalin solution. The maxilla was isolated, and the steel crowns were cut off with high-speed burs. The maxilla was separated sagittally and fixed for an additional 2 weeks in a 10% neutral formalin solution and then decalcified in 4.13% ethylene-diamine-tetracetic acid at a pH of 7.2.9 Decalcification was assessed by radiography and a chemical test with ammonia and ammonium oxalate.‘O The specimens were embedded in paraffin after orienting them in the mesiodistal plane. The treated pulpal area was then selected and cut serially at 7 pm and stained with hematoxylinphloxine-safran. All the sections passing through the

94

Jaber, Maser&,

and Donohile

pulp

necrosis

neodentin after 28

bridge days

DYCAL

Fig. 1. Drawings made with camera iucida apparatus from specimenstreated with Qsteogen and Dycal.

Fig. 2. A, Osteogen, 7 days. At left, formation of new dentin fnd] is shown projecting from dentin walls (0). B, Dycai, 7 days. At right, odontoblasts (Od) can be observed ;n association with hard tissue formation. Exposure site is shown by arrowhead. (Hematoxylin-phloxine-safran stain; original magnification, X25.)

exposure site were examined under an OrthopSan light microscope (Leitz, Germany). Pulp inflammation was evaluated according to the following scale: grade 0, normal pulp; grade 1, acute

Fig. 3. Neodentin formation (arrowhead), in Qsteogentreated specimen. This is observed around dentinal chips after 7 days in both Osteogen and Dycal specimens.Large layer of reparative dentin (RD) extends from wall of pulp chamber. (Hematoxylin-phloxine-safran stain; original magnification, X25.)

puipitis; grade 2, chronic pulpitis; grade 3, puIp necrosis (Table I). A qualitative analysis of pulpal calcification and dentin formation was also undertaken with the following scale: 0, no dentin formation; I, an incomplete dentinal bridge; C, a complete dentinal bridge (Table II). The quality of the dentin was classified as being either of a globular or tubular type. In addition, scattered calcifications throughout the pulpal chamber or the radicular pulp were noted. The dentinal bridge surface after 14 and 28 days was analyzed by a quantitative method. In each specimen three consecutive slides passing through the exposure area were selected. With the help of a camera lucida apparatus adapted to a microscope (Wild, Germany), the projection of each histologic section was traced onto a separate sheet of paper. The resultant drawings outlined the whole crown as well as the areas of necrosis and the dentinal bridge (Fig. I). The system was calibrated with a Bausch & Lomb micrometric scale, which, when projected on paper, gave a magnification factor of 83. A total of 129 drawings were made for this study. The Dycal specimens and the Osteogen specimens were drawn alternatively to reduce the effects of operator error. The 129 drawings were then submitted at random to computer-assisted automatic image analysis with a Zeiss Videoplan (Germany). The area of the dentin bridge was plotted by a person not otherwise involved in this project, with an anonymous code for the drawings. The results were automatically transformed into the real measurements of the specimens.

Dental

Volume 73 Number 1

pulp reaction

to hydroxyapatite

95

Fig. 4. A, Osteogen, 14 days. Tubular dentin formation (td) was observed with Osteogen. Same pattern was also

noted when Dycal was used. B, Details of A showing odontoblast-like cells (C)d) close to tubular dentin induced by Osteogen. C, Particles of Osteogen are encompassedby hard tissue (arrmheads). D, Details of C showing Osteogen particles. (Hematoxylin-phloxine-safran stain; original magnifications: A, X10; B, X40; C, X25; D, X40.)

Table III. Computer evaluation of dentin bridge

formation 7iizr-p-L Osteogen Dycal Osteogen Dycal

14 14 28 28

127.80 93.95 201.59 170.87

+ 78.45 * 41.39 f 109.29 -L 120.01

O.l

The width of the perforation was noted, to verify the uniformity of the surgical pulp exposures. These quantitative results were submitted to an analysis of variance. RESULTS

The pulp exposures measured on the drawings by computer showed a uniform size. The exposure sites ranged in size (mean + SD) from 260.0 f 84.1 pm to 264.6 f 95.8 /zm for the four groups, and the analysis of variance did not demonstrate any statistical difference in the diameter of the pulp exposures in the various groups (p > 0.5). This implies that the surgical trauma caused by the pulp exposures was similar in all the teeth. The dental pulps were always compared with the third molar pulp, ,which was used as a normal control. After 7 days, r-mdifference was found between the inflammatory reactions in the pulps treated with Osteogen or Dycal (Table I). An acute partial pulpitis

Fig. 5. Dycal, 14 days. Under necrotic area and inflamed pulp tissue dentinal chips (arrowheads) are incorporated in incompleted dentinal bridge. Underlying acute partial pulpitis is noted (asterisk) (Hematoxylin-phloxine-safran stain; original magnification, X 10.)

was observed in most of the pulps. Reparative dentin formation with cellular inclusions was observed extending from the lateral dentin walls in both groups (Fig. 2). Reparative dentin formation was also observed around dentinal chips at the exposure site in both groups (Fig. 3). After 14 days, the pulpal inflammation was more acute in the Osteogen-treated cases as compared with the Dycal-treated pulps, In two cases the pulps treated with Dycal showed no sign of inflammation (Table I). Dentinal healing of the exposure sites was more frequent and more extensive when Dycal was used (Table 11). Complete dentinal bridges were observed in three Dycal-treated cases, compared with only one case after the application of Osteogen. However, the dentinal bridges were thicker and more diffuse, as measured by computer, when Osteogen was used (Table III). These measurements are compatible with the qualitative observations; that is, the dentinal bridges associated with the use of Dycal were more

6

Jaber, Mascrbs, and Donahue

ORAL

5i~uKG

()IRh

.biEl,

ORAL

PWTHOL

January 1992

A, Dycai, 2% days. Dentinal bridge (nd) has been completed under exposure site (arrowheads). Details of bridge seen in A. Tubular dentin contains cellular inclusions (arrowheads). G, Qsteogen 28 days. Bridge (arrowheads) is more extensive and more heterogeneous than with Dycal. (Hematoxylin-phloxine-safran stain; original magnifications: A, X10; B, X25; 6, X10.)

localized around the exposure site than the bridges formed after the use of Osteogen. With either material the new dentin formation appeared to be of both tubular and globular types. The proportion of tubular to globular dentin was 7:9 with Dycal and 3:8 with Osteogen. In the slides examined neo-odontoblasts were frequently observed under the pulp-capping materials (Fig. 4, A and B). The dentinal debris found in the pulp near the exposure was incorporated into the new dentin bridges as the healing progressed (Fig. 5). Occasionally, some Osteogen particles were seen surrounded by new dentin (Fig. 4, C). After 28 days, inflammation was still present in the

pulps regardless of the product used. However, chronic inflammation was more frequently observed when Osteogen was used. In five pulps treated with Dycal no inflammation was present (41%) (Table I). However, our purpose was to observe the dentinal bridge and not to study the inflammatory process. Furthermore, healing of the exposure sites with new dentin occurred more frequently with Dycal (seven cases, 58%) than with Qsteogen (five cases, 38%) (Table II and Fig. 6, A and C). However, the difference in dentinai healing provoked by the two materials was not statistically significant (p > 0.1) (Table III). In all cases, regardless of the material used or the time of killing, a large area of pulp necrosis was noted

Volume 73 Number 1

in the center of the hard tissue bridge (Fig. 7). However, the necrotic areas were larger with Osteogen than with Dycal. Scattered areas of dystrophic calcifications were also noted. These areas of dystrophic calcification were more frequent when Osteogen was used (seven in the chamber, two in the radicular pulp) than when Dycal was used (two in the chamber, one in the radicular pulp). After 28 days, the kind of reparative dentin formed was equally divided between the tubular (Fig. 6, B) and the globular types. Six specimens of each type were observed in the Dycal-treated and Osteogentreated teeth. DISCUSSION

The rat is a convenient animal lo use in studies of the dental pulp. However, uniform pulp exposures require great manual dexterity even under a stereoscopic microscope, and special instruments such as lip and cheek retractors and a minialure amalgam carrier are required. A consistent problem, as reported by Rowe, 1is the difficulty in keeping the amalgam in the rat tooth cavities for an extended period of time. The steel crown technique described here is one way of protecting the pulps from the effects of leakage. By feeding the animals a ground diet, there was also less chance Iof the crowns being disturbed. After 28 days the rats appeared in good health and had shown continued weight increases. The uniform diameter of the pulp exposures, as confirmed by the computer measurements, indicated that a standardized method of pulp exposure was achieved. The pulpal inflammation observed during the experimental procedure resulted from the cumulative effects of trauma, possible bacterial contamination, and material toxicity. This may explain why the degree of pulp inflammation was similar after 7 days regardless of the material used.” However, this similarity was not evident at 14 days, when the inflammation was more diffuse after the use of Osteogen. This difference in the inflammatory reaction was even more striking after 28 days in Osteogen-treated cases, when the chronic reaction was intense and scattered throughout the tissues. This may be due to the pH of the materials. Dycal (pH 11) induces a focal necrosis when it contacts the pulp. The alkalinity of the Dycal then balances the acidity of the inflammatory exudate. On the other hand, Osteogen (pH 6.41) allows localized toxicity. Regardless of the pH of the materials, the pulp i;s able to produce neodentin as evidenced by Rowe,’ who used stannic oxide (pH 4) as a pulp-capping agent. Cox et a1.12showed that pulpal inflammation after pulp capping is caused more by the

Dental pulp reaction to hydroxyapatite

97

bacterial leakage and contamination than by the toxicity of the material. The HA particles were surrounded by new dentin. This feature could be explained by their relatively large size and their chemical composition, and was never seen with Dycal. This might explain the scattered calcifications, which were more numerous with Osteogen (three in the chamber, one in the radicular pulp) compared to Dycal (two in the chamber). HA is routinely used in implants and is considered to be osteoconductive and possibly osteoinductive under certain circumstances.13 Dentin formation can be induced in the pulp in three ways. First, dentinogenic cells can be stimulated to induce hard tissue synthesis from the dentin walls. Second, the formation of hard tissues can be provoked around the dentin chips that are pushed into the pulp during the procedure. According to Seltzer and Bender,14 this can occur spontaneously. These methods of hard tissue induction are common to both Osteogen and Dycal. Finally, hard tissue formation was observed around Osteogen particles. In contrast to Dycal, which is easy to insert into a cavity, Osteogen is made of large particles that do not allow easy manipulation. It is possible that some of them reached the deeper layers of the pulp and gave rise to diffuse calcification. In our experiment almost every pulp showed hard tissue formation, but a complete or almost complete bridging of the exposure occurred more frequently with Dycal. With this in mind, and in view of the fact that Osteogen can induce extensive dystrophic calcification in the pulp, we believe that the use of Osteogen as a pulp-capping agent in human teeth is contraindicated. The calcification of the pulp would render root access difficult if endodontic treatment were necessary later. We thank GBD Marketing Group Inc., Valley Stream, N.Y. for providing the Osteogen (HA Resorb), and Mrs. Bernadette Salnave for her help in preparing the slides used in this work. REFERENCES 1. Rowe AHR. Reaction of the rat molar pulp to various materials. Br Dent J 1967;122:291-300. 2. Paterson RC. Pulp response in sound and carious teeth: a pilot study. ORAL SURC ORAL MED ORAL PATHOL 198 1;51:209-12. 3. Chao SY, Poon CK. Histologic study of tissue response to implanted hydroxyapatite in two patients, J Oral Maxillofac Surg 1987;45:359-62. 4. Takaoka K, Nakahara H, Yoshikama H, Masuhara K, Tsuda T, Ono K. Ectopic bone induction on and in porous hydroxyapatite combined with collagen and bone morphogenetic prot&n. Clin Orthop Rel Res 1988;234:250-4. 5. Hevs DR. Cox CF. Hevs RJ. Avery JK. Histoloeical considerations of direct ‘pulp capping agents. .I Den; Res 1981; 60:1371-9. 6. Heller AL, Koenigs JF, Brilliant JD, Melfi RC, Driskell TD.

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7.

8. 9.

10. 11.

12.

Jaber, Mum&s, and Donohue Direct pulp capping of permanent teeth in primates using a resorbable form of tricalcium phosphate ceramic. J Endod ;975;3:95-101. El Deeb M, Holmes RE. Tissue response to facial contour augmentation with dense and porous hydroxylapatite in rhesus monkeys. .J Oral Maxillofac Surg 1989;47: 1282-9. Luu HT. Marrnan R, Mascrbs C. A miniature amalgam carrier. Operative Dent’1980;5:146-8. Warshawsky H, Moore G. A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 1967;15:§42-9. Culling CFA, Allison RT, Barr WT. Cellular pathology technique. 4th ed. London: Butterworths, 1985. Jaber L, Mascrts C, Donohue WB. Electron microscope characteristics of dentin repair after hydroxylapatite direct pulp capping in rats. J Oral Path01 Med (in press). Cox CF, Bergenholtz G, Heys DR, Syeol SA, Fitzgerald M,

CW.ALS~RGC~~L

"im

ORALPXTH~L January 1992

Neys RJ. Pulp capping of dental pulp mechanically exposed to oral microfiora: a 1-2 year observation of wound healing in the monkev. J Oral Path01 1985:14:156-6X. 13. Donahue WB, Mascres C. Effect of hydroxylapatite on bone formation around exposed heads of titanium implants in rabbits. J Oral Maxillofac Surg 1990$&l 196-200. 14. Seltzer S, Bender IB. The dental pulp. 3rd ed. Philadelphia: JB Lippincott, 1984. Reprint requests: C. Mascrts, DrCD, PhD Department of Stomatology Faculty of Dental Medicine University of Montreal P.O. Box 6128, Station A Montreal, Quebec Canada H3C 357

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Reaction of the dental pulp to hydroxyapatite.

The purpose of this study was to evaluate the action of hydroxyapatite (HA) (Osteogen HA Resorb, GBD Marketing Group Inc., Valley Stream, N.Y.) on the...
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