Electron microscope characteristics of dentin repair after hydroxyiapatite direct puip capping in rats
Louay Jaber\ Christiane Mascres^ and William B, Donohue^' 'Department ol Stomatology, Faculty of Dental Medicine. University of Montreal, 'Division of Orai and Maxiilo-facial Surgery, St. tvlary's Hospital Center, Montreal, Canada
Jaber L, Mascrcs C, Donohue WB: Eleetron mieroscope characteristics of dentin repair after hydroxyiapatite direct pulp capping in rats. J Oral Pathol Med 1991; 20; 502-8. In order to study the osteogenie action of hydroxyiapatite (HA) on the dental pulp, a pulp capping experiment was designed using the rat upper molar. Under general anesthesia, molar teeth in 14 male Sprague-Dawley rats were pulp capped with Osteogen (HA) or with Dycal as a control material. After pulp capping, the maxillary molars cavities were restored with amalgam and a pedodontic steel crown was adjusted and sealed over the molar teeth on either side of the maxilla. After 7 days, the areas of necrosis and acute inflammation were more evident in the pulps treated with Dyeal than with Osteogen. Hard tisstie formation began to appear around dentinal chips in the pulp and extended from the cavity walls into the pulp regardless of the material that was used. Furthermore, this calcified material was seattered throughout the pulp when Osteogen was used, but was not observed in the Dycal treated pulps. The hard tissue formation was thought to be due to the putative Tibroblasts and odontoblasts found in the pulp. After 28 days dense dentinal tissue was observed bridging the exposure site when Dycal was used. The dentinal tissues formed with Osteogen was always of a globular type, and showed an irregular distribution. Since Osteogen tends to cause areas of dystrophic calcification in the pulp, its use is not be reeommended for pulp capping purposes in humans, because these areas of calcification would make future endodontie treatment difficult.
Hydroxyiapatite (HA) is a standard material used in implantology. When used for preprosthetic reconstruction of the alveolar crest in edentulous patients with maxillary atrophy, bone apposition is sometimes observed around the hydroxyiapatite particles (1). The osleogenic potential of HA has also been tested experimentally, in tissues not normally associated with bone formation. TAKAOKA et al. demonstrated that when HA which had been previously soaked in purified bovine skin collagen was implanted in mouse muscle, it induced bone fonnation (2). However, in others studies, no bone formation was found around the hydroxyiapatite implants when used to increase deficient mandibular alveolar ridges (3), or when placed around titanium implants (4). Even if the osteogenic qualities of collagen/HA are well known, very little
researeh concerning the reaction of the dental pulp to HA have been published. Moreover, the results are often contradictory. For example, after pulpotoinies in monkeys, HA capping induced dentinal bridge formation (5). However, in the same animals, when HA was used as a direct pulp-capping agent, it failed to consistently produce complete dentinal bridges, and the dental pulp frequently showed a chronic pulpitis (6). Several studies using electron microscopy have examined the dentinal bridge after direct pulp-capping using Dycal a calcium hydroxyde containing cement (Dycal, C D . Caulk Company). As observed by scanning electron microscopy, dentinal bridges were observed in 3 human teeth after pulpotomies (7), as well as after direct pulp capping (8). To complement routine histologie observations, certain aspects of
Key words: dystrophic: calcification, dentinal bridge, Dycal: Osteogen; pulp-capping. Christiane Mascrfes, Department of Stomatoiogy, Faculty of Dental Medicine. P.O. Box 6128. Station A. Montreai, P.O. Canada, H3C 3J7 Accepted for publication June 2. 1991.
the tissue response to pulp eapping and dentinal bridge formation, were examined by electron microscopy. Previous transmission electron microscopic studies have examined the cellular response to pulp-capping using calcium hydroxyde in the incisor (9) and in molars of rats (10). However, to date, no reports have been published using the scanning electron microscope to evaluate dentinal bridge formation in rat molars after direct pulp capping. Hence, the present study was designed to examine the effeet of HA on the dental pulp of the rat molar using transmission and scanning electron microscopy. Materiai and methods Fourteen male Sprague-Dawley rats weighing between 200 and 250 g were used for this experiment. They were
HA - pulp - capping 503 housed one per cage, fed with powdered purina chow and provided with fresh tap water ad libitum. The rats were weighed once a week. Under general anesthesia induced by intraperitoneal injection of sodium pentobarbital (Somnotol; 50 mg/kg), the animals were fastened on their backs to a surgical board. The mouth was kept open with the help of a lip and cheek retractor which had been specially designed to provide access to the maxillary first and second molars. Pulp exposure and direct pulp-capping were performed on both left and right molars with the aid of a Bausch and Loinb stereoscopic microscope (magnification x 0.7 to 9) and under intermittent saline irrigation using a custom-made irrigation-aspiration apparatus. After having cleaned the molars with a cotton pellet soaked in 70% alcohol, a cavity was prepared with a tungsten 3314 inverted cone bur eonnected to a low speed handpiece. The very thin residual dentin layer above the pulp was delicately perforated with a sharp probe in an attempt to achieve pulp exposures of a consistant diameter. The width of the exposure site was latter verified histologically. The exposed pulps were dried with the smooth edge of sterile extrathin endodontic paper points. A resorbable HA product: Osteogen (HA Resorb-GBD Marketing Group Inc) was used as the experimental direct pulp-capping agent. Osteogen is composed of rod-shaped particles dissimilar in shape and volume but with a maximal length of 0.5 mm and O.I mm in width. The material is different from the resorbable tricalcium phosphate ceramic (Synthos) used by others and which has a powder-like consisteney (5-6). A calcium hydroxyde product: Dycal (L.D. Caulk Company) was used as the control material. Alternatively, Osteogen or Dycal were placed in the maxillary right or the left molars. The eavities were then filled with amalgam (Dispersalloy)
Eig. 1. Schematic diagram showing orientation of fracture plane used for preparation of teeth for seanning electron micro,seopy.
V A
\
• ' • .
o:
.
^*
•
(2B
Eig. 2. Osteogen, 7 days post operative. A, hard tissue produetion appeared to be within the pulp (arrow heads) below pulp-eapping material (D, dentin. P. pulp), (decalcified section stained with toluidine blue, x 10). B, odontoblast-like cells (arrow heads) are observed adjacent to mineralized areas (decaleified section stained with toluidine blue, x 40).
using a miniature atnalgam carrier designed for rodents (11). Pedodontic No. 6 steel crowns for human incisors were adjusted and sealed with zinc phosphate cement on the three maxillary molars of each side. The animals were anesthetized, flushed with an intraeardiae perfusion of 0,8% saline solution to remove the blood, then with a 3% glutaraldehyde solution at a pH of 7.2 using a phosphate buffer at room temperature. The maxillary molars were isolated; the steel erowns were removed with high speed burs and additional fixation was achieved by a three hour immersion in
3% glutaraldehyde. One group of 7 rats was sacrificed 7 days after the experimental procedure in order to study the cellular response by transmission electron microscopy, A second group of 7 animals was allowed to survive until 28 days after pulp capping in order to observe the dentinal bridge using the seanning electron microscope. Tissue preparation for transmission electron microscopy (TEiM)
After fixation, the teeth were dissected from the maxillary and decalcified for 4 wk in a 4% ethylene diamine tetracetic
504
JAlitiR el at.
Fig. S. Dycal, 7 days postoperative. A, hard tissue indicated hy arrow head radiates from dentinal wall (decaleified seetion stained with toluidine blue, x 10). B, hard tissue formation indicated by arrow head in A is surrounded by abundant, aetive looking eells (arrowheads) (decaleified seetion stained with toluidine blue, X 40).
acid (EDTA) solution (12) which was renewed daily. The efficacy of the decalcification was checked with a routine chemical test using ammonium oxalate for calcium detection (13). The 28 teeth, half of which were treated with Osteogen and the other half with Dycal, were then dehydrated and embedded in Epon using standard procedures. Six teeth treated with Osteogen and six teeth treated with Dycal were then selected at random for the present study. One micrometer semi-thin sections were cut in the pulp exposure area with glass knifes on a Porter Blum MTl rnicrotome. The sections were cut in the mesio-distal plane and stained with toluidine blue. Thin, gold-colored sections were cut with a diamond knife, placed on formvar-coated grids and conventionally stained with uranyl acetate and lead citrate. Sections were observed and photographed with a Philips 300 transmission electron microscope. Tissue preparation for scanning eiectron microscopy (SEM)
After fixation, the teeth were separated from the maxilla but not detnineralized. Three teeth treated with Osteogen and three teeth treated with Dycal were randomly choosen for this study. After quick freezing in 22 Freon they were immersed and stored in liquid nitrogen at — 196 C. At the time of dissection, the teeth were placed in Petri dishes containing liquid nitrogen, and with the help of a surgical blade inserted transversaly to the pulpal floor, the teeth were split open (Fig. I) and the radicular part of the teeth was discarded. The coronal part of the teeth bearing the dentinal bridges were prepared for ob-
Fig. 4. Osteogen, 7 days postoperative. Collagen-producing eell showing large nueleus (N) and abundant rough cndoplasmie retieulum (rer). Numerous eollagen fibrils (coll.) surround this eell type (deealcified section stained with uranyl and lead, TEM x 6,800).
HA - pulp - capping 505 Hard tissue neoformation was noted in the six Osteogen speeimens where it ^ was more disseminated than in the Dycal specimens (Fig. 2A shows Osteogen pattern compared to Fig. 3A Dycal). Hard tissue synthesis was seen extending from the existing walls of the pulp ehamber as well as around the dentin chips in the pulp (Fig. 2A). Areas of dystrophic calcification were also noted in the soft tissues. This hard tissue production in the pulp seems to be a unique characteristic of Osteogen pulp capping. In fact, the areas of dispersed pulpal calcification as observed in the Osteogen specimens (Fig. 2A) were not evident when Dycal was used. Nevertheless, all of the speeimens treated with Dycal, with one exception, showed initial hard tissue formation extending from the dentin walls (Figs. 3A and B) and around dentin chips. The dentin formation was less extensive with Dycal than with Osteogen. With Osteogen and Dyeal, cells with morphological similarities to odontoblasts were noted in proximity to the areas of hard tissue formation (Figs. 2B and 3B). Collagen-producing cells of comparable appearance were observed by eleetron microscopy in thin sections when Osteogen or Dycal were used. These cells had a large nucleus with heterochromatin and euchromatin homogeneously distributed. The cytoplasm contained extensive rough endoplasmic reticulum, a Golgi apparatus, and numerous small vesicles. Numerous collagen fibrils accumulated around these cells and were abutted directly against the cell membrane (Fig. 4). Similar feaEig. 5. Dycal, 7 days postoperative. .V, coUagen-piodueing cells show dense rough endoplasmie tures were observed in dental pulps treated with Dycal (Fig. 5A), Collagen reticulum (rer) (decaleified section stained with uranyl and lead, TEM x 4,500). B, dentin chips (de) are surrounded by eollagen eapsule indicated by arrow heads (decalcified section fibers on occasion formed a capsule stained with nranyl and lead, TEM x 4,500). around the dentin ehips (Fig. 5B). Twenty eight day post-operative tissue
servation by placing them for 1 h in a 30% glycerol solution in phosphate buffer at room temperature. Soft tissues were then digested by immersion for 24 h in a 12'Vi. sodium hypoehlorite solution which was renewed every 8 h. They were then washed for 6 h in distilled water which was changed every 15 min. Afterwards, they were dehydrated using standard methods and critical point dried with Peldri II (Ted Pella Inc.), following the manufacturer's instructions. Samples were examined ttsing a JEOL-JSM 840 seanning electron microscope.
response (SEM)
Results Seven day post-operafive tissue response (TEM)
Seven days after pulp capping, an area of necrosis was observed at the exposure site. Neerotic tissue was present after the application of both Osteogen and Dycal, btit was more extensive when Dyeal was used. On the other hand, areas of acute pulpitis with large numbers of neutrophilic polytnorphonuclear leucocytes were consistently observed below the areas of necrosis but were less intense and milder with Osteogen than with Dyeal.
In all three Osteogen treated samples, the dentinal bridges were not limited to the exposure site but appeared to be dispersed throughout the pulp tissue. Two dentin bridges were complete and globular in nature. In one of them, a dentin defect was evident (Figs. 6A-C). One other specimen showed an incomplete bridge of the globular type. This pattern might be attributable to rapid dentin synthesis. In the three specimens treated with Dycal the dentin bridge appeared complete and more localized around the pulp exposure sites when compared with
506
JABHR et at.
the Osteogen treated specimens (Fig. 7A). This dentinal bridge was of a globular type in two cases (Fig. 7B and C) and of a tubular type in one case.
Discussion The rat dentition is frequently used for pulp-capping studies. The biology of the rat molar pulps and the incisor pulps are different. The latter showing continuous growth and eruption while in the former, the growth parameters are similar to those seen in human teeth. Because of this similarity, we chose the rat molar instead of the incisors. The use of a steel crown as a protective device for rat molars has yet to be described in the literature. Some claim that it is impossible to keep amalgams in rat cavities for more than 30 days (14). The use of these steel crowns avoids leakage through the amalgam fillings and may extend the time these restorations can be kept in the rat molar. Very few scanning electron microscope observations have been done after pulp-capping procedures, and to the best of our knowledge, none have been published using the rat as the experimental animal. This is probably due to the technical difficulties involved. In order to evaluate the dentin formation the operator has to choose the precise point at which to fracture the frozen tooth before further observations are possible. The advantage of this technique is that it allows examination of the pulp chamber ceiling. Seven days after pulp capping, necrosis and infiammation were present as the result of trauma and irritation from the eapping agents. Areas of necrosis were observed under both Osteogen and Dycal. The difference in the intensity of necrosis and the underlying inflammation indueed by the two materials may be caused by differences in pH. Dycal is alkaline with an approximately pH of 11 and causes coagulation necrosis with an acute localized inflammatory reaction. Osteogen is slightly acidic with a pH of 6.4, and thus does not neutralize the acidity of the inflammatory fluids. The acidity of Osteogen appeared to cause a diffusion of the pulpal inflammatory proeess, which was more dispersed than when Dycal was used. The effects of the pH on pulpal inflammation has been described by SRLTZKR & Br.NDF.R (15). During recovery, the pulp as observed by these authors, produced increased calcifications. A difference in
Eig. 6. Osteogen. 2S days postoperative. A, denlinal bridge indieated by blaek arrow heads is seattered through pulp tissue (D normal dentin) (undeealeified speeimen, SEM x45). B, detail of dentin defeet shown in A with white arrow (SEM. x 500). C, details of dentin, globular type (SEM x 4,500).
the degree and extent of dystrophic calcification when these products were used, was observed. Osteogen, because of its acidity should provoke extensive areas of calcification throughout the pulp. Dycal frequently causes areas to dentinogenesis extending from the dentin wall adjacent to the exposure site toward the center of the exposure. Furthermore, Osteogen which is only available in relatively large particles, is not as easily manipulated as Dyeal. Because of this, the Osteogen particles may have been forced into the pulp and served as a nidus for dentinogenesis. The 7. day, Osteogen-treated speeimens showed pulpal calcifications more
frequently than did the Dyeal-treated pulps. When Dycal was used, a complete denlinal bridge at the site of the pulpal exposures was observed in every ease after 28 days. Technical difficulties did not allow us to confirm these results with additional specimens. Using electron micro.scopy, collagenproducing cells were observed after Osteogen or Dycal applications. These cells appeared to be inorphologieally similar to odontoblasts. This is in agreement with previous observations in humans, that neo-odontoblasts are formed after pulp capping with calcium hydroxyde (15). Odontoblasts are generally considered as being tubular dentin pro-
HA - pulp - capping 507 .icknowtedgments ~ We wish to thank GBD Marketing Group Ine. for providing the Osteogen (HA Resorb TM). We also wish to acknowledge the help and cooperations of Drs. ANTON'IO NANCI and MARC MCKEE, and Ms BERNADETTE SALNAVE for her help in pre-
paring sections for transmission electron miero.seopy, and Ms SYLVIA ZALZAL for SEM
speeimens. References 1. CHAO SY, POON CK. Histologic study of
tissue response to implanted hydroxyiapatite in two patients. J Oral Maxillofac Surg 1987; 45: 359-62. 2.
TAKAOK.A K , N A K A H A R A
H , YOSHIKAWA
H, MASUHARA K , TSLIDA T, ONO K . Ec-
topic bone induction on and in porous hydro.xylapatite combined with eollagen and bone morphogenetie protein. Clin Orthop Rel Res 1988,- 234: 250-4. 3. BEIRM: O R , GREENSPAN JS. Histologie
evaluation of tissue response to hydroxyiapatite implanted on human mandibles. ./ 0.71/ Res 1985; 64(9): 1152-1154. 4. DONOHUE WB, MASCRES C . Effect of hy-
droxyiapatite on bone formation around exposed heads of titanium implants in rabbits. J Oral Maxittofac Surg 1990; 48; 1196-1200. 5. HELLER AL, KOENIGS JE, BRILLIANT JD,
MELEI RC. DRISKELL TD. Direct pulp
eapping of permanent teeth in primates using a resorbable form of tricalcium phosphale eeramie. J Endod 1975; 3; 95-101. 6. HEVS DR, Cox CE, HEYS RJ, AVERY JK.
Histological considerations of direct pulp eapping agents. J Dent Res 1981; 60; 1371-9. 7. ULMANSKY M . SELA J. SELA M . Seanning
electron microscopy of calcium hydroxide induced bridges. J. Orat Pathot 1972; 1; 244-8. 8. ERANZ F E , HOLTZ J, BALIME LJ. Etude
fl.?. 7. Dyeal, 28 days postoperative. A, eomplete dentinal bridge is marked by arrow heids (D, normal dentin) (undeealeified speeimen. SEM x 35). B, dentin bridge shown in A is eomposed of globular type dentin (arrow) whieh is in elose proximity to tubular dentin of wall ol tooth (SEM x 1,300). C, globular neodentin from the dentinal bridge as seen in Fia 7A (SEM X 4,500). ''•
histologique et au M.E.B. de la neoformation du pont de dentine apres coinage pulpaire direct sur dents humaines. Inf Dent 1986; 15; 1289-1304. 9. HARROP TJ. MACKAY B. Eleetron miero-
ducing cells. Globular dentin without tubules, as observed frequently in our experiments, could be produced by pulp fibroblasts which become active during pulp repair. These two different methods of producing a dentinal bridge after pulp capping allows us to assume a dual form of tissue formation involving two different kinds of cells, putative neoodontoblasts and fibroblasts. This has already been described in the literature (16). The pattern we observed in almost every case, showing a predominance of globular dentin as compared to the tubular form, is probably due to fibroblastie activity (17).
Even if Osteogen has an osteoconductive potential, it should be used with caution as a pulp capping agent. First, the size of the rod-shaped particles available for implantology is too large for pulp eapping purposes. The o"steogen adapts and adheres poorly to the cavity walls, so that inadvertently Osteogen may be forced into the pulp tissues. As was ob.served in our 7-day specimens this ean lead to areas of dystrophic calcification, which might cause problems at a latter date if endodontics should be necessary.
seopie ob.servations on healing in dental pulp in the rat. Arch Oral Biol 1968; 13; 365-85. 10. SELA J, TAMARI I, HIRSCHFELD Z , BAB
I. Transmission eleclron microscopy of reparative dentin in rat molar pulps. Aeta Atutt 1981; 109; 247-51. 11. Luu HT, MAGNAN
R, MASCR^
C. A
miniature amalgam carrier. Oper Dent 1980; 5; 146-8. 12. WARSHAW'SKY H , M(X)RE G . A technique
for the fixation and deealcification of rat ineisors for eleetron mieroseopy. J Histoehem Cylochem 1967; 15; 542-9. 13. CULLING C E A , ALLISON RT, BARR W T -
Ccttutar pathotogy technique. 4th ed. London; Butterworths, 1985. 14. RowE AHR. Reaction of the rat molar pulp to various materials. Br Dent J 1967; 122; 291-300.
508
JABER et al.
15, SELTZER S, BENDER IB, The dental pulp.
Biologic considerations in dentat procedures. Philadelphia & Montreal; J, B, Lippincott Co,, 1984,
16, HORSTED P, E L ATTAR K , LANGELAND
17, JABER L , MASCKfis C, DONOHUE W B ,
K, Capping of monkey pulps with Dycal and Ca-Eugenol cement. Oral Surg Oral Med Oral Pathoi 1981:52: 531-53,
The reaction of the dental pulp to hydroxylapatite (HA), Oral Surg Oral Med Oral Pathol (in press),
Louay Jaber\ Christiane Mascres^ and William B, Donohue^' 'Department ol Stomatology, Faculty of Dental Medicine. University of Montreal, 'Division of Orai and Maxiilo-facial Surgery, St. tvlary's Hospital Center, Montreal, Canada
Jaber L, Mascrcs C, Donohue WB: Eleetron mieroscope characteristics of dentin repair after hydroxyiapatite direct pulp capping in rats. J Oral Pathol Med 1991; 20; 502-8. In order to study the osteogenie action of hydroxyiapatite (HA) on the dental pulp, a pulp capping experiment was designed using the rat upper molar. Under general anesthesia, molar teeth in 14 male Sprague-Dawley rats were pulp capped with Osteogen (HA) or with Dycal as a control material. After pulp capping, the maxillary molars cavities were restored with amalgam and a pedodontic steel crown was adjusted and sealed over the molar teeth on either side of the maxilla. After 7 days, the areas of necrosis and acute inflammation were more evident in the pulps treated with Dyeal than with Osteogen. Hard tisstie formation began to appear around dentinal chips in the pulp and extended from the cavity walls into the pulp regardless of the material that was used. Furthermore, this calcified material was seattered throughout the pulp when Osteogen was used, but was not observed in the Dycal treated pulps. The hard tissue formation was thought to be due to the putative Tibroblasts and odontoblasts found in the pulp. After 28 days dense dentinal tissue was observed bridging the exposure site when Dycal was used. The dentinal tissues formed with Osteogen was always of a globular type, and showed an irregular distribution. Since Osteogen tends to cause areas of dystrophic calcification in the pulp, its use is not be reeommended for pulp capping purposes in humans, because these areas of calcification would make future endodontie treatment difficult.
Hydroxyiapatite (HA) is a standard material used in implantology. When used for preprosthetic reconstruction of the alveolar crest in edentulous patients with maxillary atrophy, bone apposition is sometimes observed around the hydroxyiapatite particles (1). The osleogenic potential of HA has also been tested experimentally, in tissues not normally associated with bone formation. TAKAOKA et al. demonstrated that when HA which had been previously soaked in purified bovine skin collagen was implanted in mouse muscle, it induced bone fonnation (2). However, in others studies, no bone formation was found around the hydroxyiapatite implants when used to increase deficient mandibular alveolar ridges (3), or when placed around titanium implants (4). Even if the osteogenic qualities of collagen/HA are well known, very little
researeh concerning the reaction of the dental pulp to HA have been published. Moreover, the results are often contradictory. For example, after pulpotoinies in monkeys, HA capping induced dentinal bridge formation (5). However, in the same animals, when HA was used as a direct pulp-capping agent, it failed to consistently produce complete dentinal bridges, and the dental pulp frequently showed a chronic pulpitis (6). Several studies using electron microscopy have examined the dentinal bridge after direct pulp-capping using Dycal a calcium hydroxyde containing cement (Dycal, C D . Caulk Company). As observed by scanning electron microscopy, dentinal bridges were observed in 3 human teeth after pulpotomies (7), as well as after direct pulp capping (8). To complement routine histologie observations, certain aspects of
Key words: dystrophic: calcification, dentinal bridge, Dycal: Osteogen; pulp-capping. Christiane Mascrfes, Department of Stomatoiogy, Faculty of Dental Medicine. P.O. Box 6128. Station A. Montreai, P.O. Canada, H3C 3J7 Accepted for publication June 2. 1991.
the tissue response to pulp eapping and dentinal bridge formation, were examined by electron microscopy. Previous transmission electron microscopic studies have examined the cellular response to pulp-capping using calcium hydroxyde in the incisor (9) and in molars of rats (10). However, to date, no reports have been published using the scanning electron microscope to evaluate dentinal bridge formation in rat molars after direct pulp capping. Hence, the present study was designed to examine the effeet of HA on the dental pulp of the rat molar using transmission and scanning electron microscopy. Materiai and methods Fourteen male Sprague-Dawley rats weighing between 200 and 250 g were used for this experiment. They were
HA - pulp - capping 503 housed one per cage, fed with powdered purina chow and provided with fresh tap water ad libitum. The rats were weighed once a week. Under general anesthesia induced by intraperitoneal injection of sodium pentobarbital (Somnotol; 50 mg/kg), the animals were fastened on their backs to a surgical board. The mouth was kept open with the help of a lip and cheek retractor which had been specially designed to provide access to the maxillary first and second molars. Pulp exposure and direct pulp-capping were performed on both left and right molars with the aid of a Bausch and Loinb stereoscopic microscope (magnification x 0.7 to 9) and under intermittent saline irrigation using a custom-made irrigation-aspiration apparatus. After having cleaned the molars with a cotton pellet soaked in 70% alcohol, a cavity was prepared with a tungsten 3314 inverted cone bur eonnected to a low speed handpiece. The very thin residual dentin layer above the pulp was delicately perforated with a sharp probe in an attempt to achieve pulp exposures of a consistant diameter. The width of the exposure site was latter verified histologically. The exposed pulps were dried with the smooth edge of sterile extrathin endodontic paper points. A resorbable HA product: Osteogen (HA Resorb-GBD Marketing Group Inc) was used as the experimental direct pulp-capping agent. Osteogen is composed of rod-shaped particles dissimilar in shape and volume but with a maximal length of 0.5 mm and O.I mm in width. The material is different from the resorbable tricalcium phosphate ceramic (Synthos) used by others and which has a powder-like consisteney (5-6). A calcium hydroxyde product: Dycal (L.D. Caulk Company) was used as the control material. Alternatively, Osteogen or Dycal were placed in the maxillary right or the left molars. The eavities were then filled with amalgam (Dispersalloy)
Eig. 1. Schematic diagram showing orientation of fracture plane used for preparation of teeth for seanning electron micro,seopy.
V A
\
• ' • .
o:
.
^*
•
(2B
Eig. 2. Osteogen, 7 days post operative. A, hard tissue produetion appeared to be within the pulp (arrow heads) below pulp-eapping material (D, dentin. P. pulp), (decalcified section stained with toluidine blue, x 10). B, odontoblast-like cells (arrow heads) are observed adjacent to mineralized areas (decaleified section stained with toluidine blue, x 40).
using a miniature atnalgam carrier designed for rodents (11). Pedodontic No. 6 steel crowns for human incisors were adjusted and sealed with zinc phosphate cement on the three maxillary molars of each side. The animals were anesthetized, flushed with an intraeardiae perfusion of 0,8% saline solution to remove the blood, then with a 3% glutaraldehyde solution at a pH of 7.2 using a phosphate buffer at room temperature. The maxillary molars were isolated; the steel erowns were removed with high speed burs and additional fixation was achieved by a three hour immersion in
3% glutaraldehyde. One group of 7 rats was sacrificed 7 days after the experimental procedure in order to study the cellular response by transmission electron microscopy, A second group of 7 animals was allowed to survive until 28 days after pulp capping in order to observe the dentinal bridge using the seanning electron microscope. Tissue preparation for transmission electron microscopy (TEiM)
After fixation, the teeth were dissected from the maxillary and decalcified for 4 wk in a 4% ethylene diamine tetracetic
504
JAlitiR el at.
Fig. S. Dycal, 7 days postoperative. A, hard tissue indicated hy arrow head radiates from dentinal wall (decaleified seetion stained with toluidine blue, x 10). B, hard tissue formation indicated by arrow head in A is surrounded by abundant, aetive looking eells (arrowheads) (decaleified seetion stained with toluidine blue, X 40).
acid (EDTA) solution (12) which was renewed daily. The efficacy of the decalcification was checked with a routine chemical test using ammonium oxalate for calcium detection (13). The 28 teeth, half of which were treated with Osteogen and the other half with Dycal, were then dehydrated and embedded in Epon using standard procedures. Six teeth treated with Osteogen and six teeth treated with Dycal were then selected at random for the present study. One micrometer semi-thin sections were cut in the pulp exposure area with glass knifes on a Porter Blum MTl rnicrotome. The sections were cut in the mesio-distal plane and stained with toluidine blue. Thin, gold-colored sections were cut with a diamond knife, placed on formvar-coated grids and conventionally stained with uranyl acetate and lead citrate. Sections were observed and photographed with a Philips 300 transmission electron microscope. Tissue preparation for scanning eiectron microscopy (SEM)
After fixation, the teeth were separated from the maxilla but not detnineralized. Three teeth treated with Osteogen and three teeth treated with Dycal were randomly choosen for this study. After quick freezing in 22 Freon they were immersed and stored in liquid nitrogen at — 196 C. At the time of dissection, the teeth were placed in Petri dishes containing liquid nitrogen, and with the help of a surgical blade inserted transversaly to the pulpal floor, the teeth were split open (Fig. I) and the radicular part of the teeth was discarded. The coronal part of the teeth bearing the dentinal bridges were prepared for ob-
Fig. 4. Osteogen, 7 days postoperative. Collagen-producing eell showing large nueleus (N) and abundant rough cndoplasmie retieulum (rer). Numerous eollagen fibrils (coll.) surround this eell type (deealcified section stained with uranyl and lead, TEM x 6,800).
HA - pulp - capping 505 Hard tissue neoformation was noted in the six Osteogen speeimens where it ^ was more disseminated than in the Dycal specimens (Fig. 2A shows Osteogen pattern compared to Fig. 3A Dycal). Hard tissue synthesis was seen extending from the existing walls of the pulp ehamber as well as around the dentin chips in the pulp (Fig. 2A). Areas of dystrophic calcification were also noted in the soft tissues. This hard tissue production in the pulp seems to be a unique characteristic of Osteogen pulp capping. In fact, the areas of dispersed pulpal calcification as observed in the Osteogen specimens (Fig. 2A) were not evident when Dycal was used. Nevertheless, all of the speeimens treated with Dycal, with one exception, showed initial hard tissue formation extending from the dentin walls (Figs. 3A and B) and around dentin chips. The dentin formation was less extensive with Dycal than with Osteogen. With Osteogen and Dyeal, cells with morphological similarities to odontoblasts were noted in proximity to the areas of hard tissue formation (Figs. 2B and 3B). Collagen-producing cells of comparable appearance were observed by eleetron microscopy in thin sections when Osteogen or Dycal were used. These cells had a large nucleus with heterochromatin and euchromatin homogeneously distributed. The cytoplasm contained extensive rough endoplasmic reticulum, a Golgi apparatus, and numerous small vesicles. Numerous collagen fibrils accumulated around these cells and were abutted directly against the cell membrane (Fig. 4). Similar feaEig. 5. Dycal, 7 days postoperative. .V, coUagen-piodueing cells show dense rough endoplasmie tures were observed in dental pulps treated with Dycal (Fig. 5A), Collagen reticulum (rer) (decaleified section stained with uranyl and lead, TEM x 4,500). B, dentin chips (de) are surrounded by eollagen eapsule indicated by arrow heads (decalcified section fibers on occasion formed a capsule stained with nranyl and lead, TEM x 4,500). around the dentin ehips (Fig. 5B). Twenty eight day post-operative tissue
servation by placing them for 1 h in a 30% glycerol solution in phosphate buffer at room temperature. Soft tissues were then digested by immersion for 24 h in a 12'Vi. sodium hypoehlorite solution which was renewed every 8 h. They were then washed for 6 h in distilled water which was changed every 15 min. Afterwards, they were dehydrated using standard methods and critical point dried with Peldri II (Ted Pella Inc.), following the manufacturer's instructions. Samples were examined ttsing a JEOL-JSM 840 seanning electron microscope.
response (SEM)
Results Seven day post-operafive tissue response (TEM)
Seven days after pulp capping, an area of necrosis was observed at the exposure site. Neerotic tissue was present after the application of both Osteogen and Dycal, btit was more extensive when Dyeal was used. On the other hand, areas of acute pulpitis with large numbers of neutrophilic polytnorphonuclear leucocytes were consistently observed below the areas of necrosis but were less intense and milder with Osteogen than with Dyeal.
In all three Osteogen treated samples, the dentinal bridges were not limited to the exposure site but appeared to be dispersed throughout the pulp tissue. Two dentin bridges were complete and globular in nature. In one of them, a dentin defect was evident (Figs. 6A-C). One other specimen showed an incomplete bridge of the globular type. This pattern might be attributable to rapid dentin synthesis. In the three specimens treated with Dycal the dentin bridge appeared complete and more localized around the pulp exposure sites when compared with
506
JABHR et at.
the Osteogen treated specimens (Fig. 7A). This dentinal bridge was of a globular type in two cases (Fig. 7B and C) and of a tubular type in one case.
Discussion The rat dentition is frequently used for pulp-capping studies. The biology of the rat molar pulps and the incisor pulps are different. The latter showing continuous growth and eruption while in the former, the growth parameters are similar to those seen in human teeth. Because of this similarity, we chose the rat molar instead of the incisors. The use of a steel crown as a protective device for rat molars has yet to be described in the literature. Some claim that it is impossible to keep amalgams in rat cavities for more than 30 days (14). The use of these steel crowns avoids leakage through the amalgam fillings and may extend the time these restorations can be kept in the rat molar. Very few scanning electron microscope observations have been done after pulp-capping procedures, and to the best of our knowledge, none have been published using the rat as the experimental animal. This is probably due to the technical difficulties involved. In order to evaluate the dentin formation the operator has to choose the precise point at which to fracture the frozen tooth before further observations are possible. The advantage of this technique is that it allows examination of the pulp chamber ceiling. Seven days after pulp capping, necrosis and infiammation were present as the result of trauma and irritation from the eapping agents. Areas of necrosis were observed under both Osteogen and Dycal. The difference in the intensity of necrosis and the underlying inflammation indueed by the two materials may be caused by differences in pH. Dycal is alkaline with an approximately pH of 11 and causes coagulation necrosis with an acute localized inflammatory reaction. Osteogen is slightly acidic with a pH of 6.4, and thus does not neutralize the acidity of the inflammatory fluids. The acidity of Osteogen appeared to cause a diffusion of the pulpal inflammatory proeess, which was more dispersed than when Dycal was used. The effects of the pH on pulpal inflammation has been described by SRLTZKR & Br.NDF.R (15). During recovery, the pulp as observed by these authors, produced increased calcifications. A difference in
Eig. 6. Osteogen. 2S days postoperative. A, denlinal bridge indieated by blaek arrow heads is seattered through pulp tissue (D normal dentin) (undeealeified speeimen, SEM x45). B, detail of dentin defeet shown in A with white arrow (SEM. x 500). C, details of dentin, globular type (SEM x 4,500).
the degree and extent of dystrophic calcification when these products were used, was observed. Osteogen, because of its acidity should provoke extensive areas of calcification throughout the pulp. Dycal frequently causes areas to dentinogenesis extending from the dentin wall adjacent to the exposure site toward the center of the exposure. Furthermore, Osteogen which is only available in relatively large particles, is not as easily manipulated as Dyeal. Because of this, the Osteogen particles may have been forced into the pulp and served as a nidus for dentinogenesis. The 7. day, Osteogen-treated speeimens showed pulpal calcifications more
frequently than did the Dyeal-treated pulps. When Dycal was used, a complete denlinal bridge at the site of the pulpal exposures was observed in every ease after 28 days. Technical difficulties did not allow us to confirm these results with additional specimens. Using electron micro.scopy, collagenproducing cells were observed after Osteogen or Dycal applications. These cells appeared to be inorphologieally similar to odontoblasts. This is in agreement with previous observations in humans, that neo-odontoblasts are formed after pulp capping with calcium hydroxyde (15). Odontoblasts are generally considered as being tubular dentin pro-
HA - pulp - capping 507 .icknowtedgments ~ We wish to thank GBD Marketing Group Ine. for providing the Osteogen (HA Resorb TM). We also wish to acknowledge the help and cooperations of Drs. ANTON'IO NANCI and MARC MCKEE, and Ms BERNADETTE SALNAVE for her help in pre-
paring sections for transmission electron miero.seopy, and Ms SYLVIA ZALZAL for SEM
speeimens. References 1. CHAO SY, POON CK. Histologic study of
tissue response to implanted hydroxyiapatite in two patients. J Oral Maxillofac Surg 1987; 45: 359-62. 2.
TAKAOK.A K , N A K A H A R A
H , YOSHIKAWA
H, MASUHARA K , TSLIDA T, ONO K . Ec-
topic bone induction on and in porous hydro.xylapatite combined with eollagen and bone morphogenetie protein. Clin Orthop Rel Res 1988,- 234: 250-4. 3. BEIRM: O R , GREENSPAN JS. Histologie
evaluation of tissue response to hydroxyiapatite implanted on human mandibles. ./ 0.71/ Res 1985; 64(9): 1152-1154. 4. DONOHUE WB, MASCRES C . Effect of hy-
droxyiapatite on bone formation around exposed heads of titanium implants in rabbits. J Oral Maxittofac Surg 1990; 48; 1196-1200. 5. HELLER AL, KOENIGS JE, BRILLIANT JD,
MELEI RC. DRISKELL TD. Direct pulp
eapping of permanent teeth in primates using a resorbable form of tricalcium phosphale eeramie. J Endod 1975; 3; 95-101. 6. HEVS DR, Cox CE, HEYS RJ, AVERY JK.
Histological considerations of direct pulp eapping agents. J Dent Res 1981; 60; 1371-9. 7. ULMANSKY M . SELA J. SELA M . Seanning
electron microscopy of calcium hydroxide induced bridges. J. Orat Pathot 1972; 1; 244-8. 8. ERANZ F E , HOLTZ J, BALIME LJ. Etude
fl.?. 7. Dyeal, 28 days postoperative. A, eomplete dentinal bridge is marked by arrow heids (D, normal dentin) (undeealeified speeimen. SEM x 35). B, dentin bridge shown in A is eomposed of globular type dentin (arrow) whieh is in elose proximity to tubular dentin of wall ol tooth (SEM x 1,300). C, globular neodentin from the dentinal bridge as seen in Fia 7A (SEM X 4,500). ''•
histologique et au M.E.B. de la neoformation du pont de dentine apres coinage pulpaire direct sur dents humaines. Inf Dent 1986; 15; 1289-1304. 9. HARROP TJ. MACKAY B. Eleetron miero-
ducing cells. Globular dentin without tubules, as observed frequently in our experiments, could be produced by pulp fibroblasts which become active during pulp repair. These two different methods of producing a dentinal bridge after pulp capping allows us to assume a dual form of tissue formation involving two different kinds of cells, putative neoodontoblasts and fibroblasts. This has already been described in the literature (16). The pattern we observed in almost every case, showing a predominance of globular dentin as compared to the tubular form, is probably due to fibroblastie activity (17).
Even if Osteogen has an osteoconductive potential, it should be used with caution as a pulp capping agent. First, the size of the rod-shaped particles available for implantology is too large for pulp eapping purposes. The o"steogen adapts and adheres poorly to the cavity walls, so that inadvertently Osteogen may be forced into the pulp tissues. As was ob.served in our 7-day specimens this ean lead to areas of dystrophic calcification, which might cause problems at a latter date if endodontics should be necessary.
seopie ob.servations on healing in dental pulp in the rat. Arch Oral Biol 1968; 13; 365-85. 10. SELA J, TAMARI I, HIRSCHFELD Z , BAB
I. Transmission eleclron microscopy of reparative dentin in rat molar pulps. Aeta Atutt 1981; 109; 247-51. 11. Luu HT, MAGNAN
R, MASCR^
C. A
miniature amalgam carrier. Oper Dent 1980; 5; 146-8. 12. WARSHAW'SKY H , M(X)RE G . A technique
for the fixation and deealcification of rat ineisors for eleetron mieroseopy. J Histoehem Cylochem 1967; 15; 542-9. 13. CULLING C E A , ALLISON RT, BARR W T -
Ccttutar pathotogy technique. 4th ed. London; Butterworths, 1985. 14. RowE AHR. Reaction of the rat molar pulp to various materials. Br Dent J 1967; 122; 291-300.
508
JABER et al.
15, SELTZER S, BENDER IB, The dental pulp.
Biologic considerations in dentat procedures. Philadelphia & Montreal; J, B, Lippincott Co,, 1984,
16, HORSTED P, E L ATTAR K , LANGELAND
17, JABER L , MASCKfis C, DONOHUE W B ,
K, Capping of monkey pulps with Dycal and Ca-Eugenol cement. Oral Surg Oral Med Oral Pathoi 1981:52: 531-53,
The reaction of the dental pulp to hydroxylapatite (HA), Oral Surg Oral Med Oral Pathol (in press),
