Endod Dent Traumatol 1993; V: 165-170 Prinled in Denmark . At!

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Munksgaard

1993

Endodontics & Dental Traumatology

ISSN 0109-2502

Bio-microscopical observation of dystrophic calcification induced by calcium hydroxide

Wakabayashi H, Horikawa M, Funato A, Onodera A, Matsumoto K. Bio-microscopical observation of dystrophic calcification induced by calcium hydroxide. Endod Dent Traumatol 1993; 9; 165-170. Munksgaard, 1993. Abstract - Calcium hydroxide paste was introduced into a rabbit ear chamber, and the eflect of calcium hydroxide on the vascular tissue was observed under a biomicroscope continuously up to 14 weeks. Many precipitates were observed at the border of living tissue, after the dissolution of microvessels around the paste. They increased both in size and number to combine like a river bank in the first 48 h. After 1 week, microcirculation recovered, and newly-formed capillaries approached the precipitatebank, the edge of which became smoother as weeks went by. The bank was stable and compatible to microvessels during the observation period. By SEM observation, the width of the bank was from 200 to 400 ^im, and the tissue-side of the bank appeared like amorphous lamellae, while the inner-side showed particlelike appearance. EDX examination revealed high peaks of Ca and P similar to calcium phosphate at the tissue-side of the bank, but only Ca peak at the inner-side. The precipitates seemed to have (he potential to induce dystrophic calcification by absorbing Ca and P from the tissue. Hajime Waksbavashi, Manami Horikawa, Akiyoshi Funato, Atsushi Onodera, Koukichi Matsumoto
Department of Endodontics, Showa University Sctiooi ot Dentistry, Tokyo, Japan,

Key words: calcium hydroxide: dystrophic caicificatlon; rabbit ear chamber, Hajime V^fekabayashi, Department of EndoContics, Stiowa University School ot Dentistry, 2-1-1 Kitasenzoku Ohta-ku Tokyo 145 Japan, Accepted January 13,1993

The unique potential of calcium hydroxide to induce hard tissue formation in the dental pulp and periodontium has been demonstrated by many clinical and basic experiments (1, 2). Calcium hydroxide can also induce dystrophic calcification even in the subcutaneous connective tissue that has not been programmed to mineralize (3). The latter was investigated (4-6) for the key of the bio-chemical effect of calcium hydroxide, because it was assumed that the same reaction occurs in the initial stage both in the dental pulp and in the other connective tissues. However, the mechanism is not clear so far. Most of the previous studies were done by histopathological methods, in which continuous observation in the same tissue was impossible. Therefore, it is difficult to know actually the beginning and developing of calcification in the tissue. If a continuous in vivo observation were possible, new aspects of the effect of calcium hydroxide might be discovered. The rabbit ear chamber provides a valuable tech-

nique for in vivo observation of living vascular tissue. A transparent chamber is inserted into a rabbit's ear, and after a thin vascular tissue regenerates in the chamber, a microscopic view of living microcirculation can be observed continuously in the same tissue over weeks and months without anesthesia. This technique was originated by Sandison and developed by Clerk & Clerk for the microcirculation research in 1930'. Then, various modifications of chamber design were made and adopted for experimental pathology. In dentistry, Howden et al. (7) apphed this technique to examine microcireulatory reactions to endodontic and restorative materials. However, calcium hydroxide has not been tested. Before the present experiment, the authors designed an improved rabbit ear chamber to estimate irritation potential of root canal sealers, and applied it successfully. The purpose of this study was to elucidate the mechanism of dystrophic calcification induced by 165

Wakabayashi at al. calcium hydroxide in the connective tissue, using the improved rabbit ear chamber. Interactions between microvessels and caleium hydroxide were observed under a biomieroscope immediately after the application and continuously up to 14 weeks. Then, calcified products in the chamber were observed with a scanning electron microscope (SEM), and examined for an ultimate analysis using an energy dispersion X-ray microanalyzer (EDX). Material and methods The improved rabbit ear chamber consisted of a disk and a holder ring made of transparent acrylic resin, with a mica window and a teflon plug (Fig. 1). The basic design followed Ahern's model (8), and the main modification was a well next to the observation table, through which materials could be introduced into the chamber. The well was usually obstructed by a teflon plug from the backside. Tissue spaee on the observation table was regulated to be 50 |im by a central stop, which allowed only one layer of vascular network after regeneration. One male albino-rabbit (weighing 3.0 kg) was fixed in an experimental container, and local infiltration anesthesia was given at the base of the ear. Then, one center hole and three small holes around it were punched on the upper portion of the ear with a specially-designed punch. The skin around the center hole was peeled off, leaving vascular network on the cartilage as intact as possible. After that, the chamber disk was inserted, fixed and glued to a holder ring with an adhesive agent. The space in the chamber filled with blood, and healing of the wound progressed. Microvessels were completely regenerated on the observation table usually 5 weeks after the operation (Fig. 2). Eight chambers of 7 rabbits with complete regeneration of vascular network were provided. Calcium hydroxide powder was mixed with saline (1.5 g/ml, pH 12.5) to a paste consistency used in the clinic. The teflon plug was carefully removed in an aseptic way. Calcium hydroxide paste was applied on the tip of the tefion plug, and then the plug was put back into the well to introduce calcium hydroxide to the thin vascular tissue on the observation table. The tip of the removed plug had been cut about 0.5 mm short before replacing to make the volume of the material about 1.5 mm cube. Interaction between the material and the microcirculation inside was observed continuously under a biomicroscope, immediately after the application, after 3, 6, 12, 24, 48, 72 h, after 5 days, then, every week up to 14 weeks. A light microscope equipped with a special observation stage and a camera was used with magnification from 10 to 400. At three different periods of 24 h, 1 week and 14 weeks after the application, two chambers each were removed under a local anesthesia. The observation tables with precipitate products were taken out and prepared for SEM and EDX. They were air-dried, sputter-coated with carbon and examined with a SEM 'JOEL T220A; accelerating voltage: 20 KV). Then, ultimate analysis of the precipitate products

Eig, I, Design of the improved rabbit ear chamber (length: mm] A: Disk, B: Holder ring, C: Mica window, a; pillar, b: central stop, c: observation table, d: well.

Fig, 2. Illustration of the sectioned chamber, a: vascular tissue, b: inserted material, c: regenerated tissue on the observation table, d: ear of a rabbit, e: cartilage.

Eig, 3. Observation table '^ h after the application. A: Microcireulatory network, B: Precipitates, C: Well (calcium hydroxide paste), *: central stop.

186

Dystrophic calcification by Ca|OH),

on the table were carried out mainly on Ca and P elements with an EDX (Kevek DELTA IV; accelerating voltage: 20 KV, specimen current: 2x 10"'" A, count time: 200 sec). The vascular tissue on the observation table was cut off at the same time, fixed with 10% formalin solution and proceeded in the routine manner of histopathological investigation.
Resolts Biomicroscopic observation

Several minutes after the application of calcium hydroxide, microvessels within close vicinity to the paste began to cause stasis, then disappeared in succession. A zone of tissue dissolution appeared concentrically around the well with the paste within an hour. After 3 h, minute precipitates were observed at the border between the living vascular tissue and the dissolved area (Fig. 3). Microcirculalion on the observation table showed mild disorders caused by the tissue dissolution, such as stasis, slugging and adherence of leucocytes. From 6 to 48 h, the precipitates were increasing both in size and number. They grew by fusing and combining with each other, from many string-like shaped pieces to belt-like ones, finally appeared like a river bank concentrically around the well (Figs. 4, :")). After 72 h, the rapid formation of precipitateproducts ceased, and microcirculatory disorders became less and less. The width of the bank varied among the specimens from 100 to 200 ^m. Inside the precipitate-bank, a zone of tissue defect with necrotic debris was still observed as a light brown transparent zone. One week after the application, microcirculation had almost recovered from disorders. The precipitate-bank appeared to be a substantial barrier for the living vascular tissue. On the tissue-side of the

bank, newly-formed capillaries were growing into the precipitates (Fig. 6). No pathological features were noticed in their form and function, except slight leucocyte adherence remaining up to 7 weeks. From 2 to 14 weeks, the findings on the observation table were constant without any specific changes to be mentioned. The precipitate-bank revealed good compatibility with the connective tissue and microcirculation, being stable without disintegration or resorption. The edge of the bank on the tissue-side appeared to change gradually into an amorphous and smooth form, while on the well-side of the bank the precipitates remained particle-like in form (Fig. 7).
SEM and EDX examination

It was found that all the precipitate products in the chamber firmly adhered to the surface of the observation table. SEM observation of 24-h specimens revealed that the precipitates were in form of

Fig. 5. Observation table 48 h after the application. A: Microcirculatory network, B: Precipitates, C: Well (calcium hydroxide paste), *: central stop.

Fg. 4. (.)bKerv;iii(Mi table 12 h after the application. A: Microcirfulatory network, B: Prctipitates, C: Well (calcium hydroxide |)iiste), *: central stop.

Fig. 6. Newly-formed capillaries and precipitates 2 weeks after the application. A: growing capillaries, B: precipitates.

167

Wakabayashi et ai.

Fig. 7. Observation table 7 weeks after the application. A: Microcirculatory network, B; Precipitates, C; Well (calcium hydroxide paste), *: central stop.

Fig. 9. SEM findings of the precipitate-products 'i4- h alter the application ( x 150), A: tissue-side, B: well-side.

Fig. 8. SEM tindiii: s o\' the obser\'atinn table 24 li after application ( x ir>Oj a: (altium t.arbonate-like precipitate; b: fused precipitates; c: well (calcium hydroxide paste).

?; 1(1 S L M tiiidui^'sot V lUOi r>'stab. A tibsue-sidt,

j.nni I M WH ks alt( i the appiu .itinn uell-side, a btc-hne-shaped ].ii^i

calcite of 20 fim in diameter, which resembled the crystals of calcium carbonate (Fig. 8). This crystal itself showed only a high Ca-peak by EDX analysis. These crystals fused to larger clusters here and there on the observation table around the well. Amorphous deposits between fused precipitates showed a lower Ca-peak and also weak peaks of S and P. In 1-week speciments, the precipitated bank was over 200 |im wide. Fusion of the crystals advanced more on the tissue-side of the bank (Fig. 9). Only a high Ca-peak was detected from single crystals at the well-side, whereas a medium Ca-peak and a low P-peak were detected in the middle portion of the bank. In some areas, weak peaks of S and Mg were also found. On the tissue-side, both high Ca- and high P-peaks similar to calcium phosphate were detected. The Ca and P came from the tissue and not from the material in the well. In 14-week specimens, the bank was over 400 (im wide (Fig. 10). Morphlogy and element component 168

of the bank were different from portion to portion. On the well-side of the bank, precipitates became considerably larger in size, but less in number. Beehive shaped large crystals about 100 |im in diameter were found on the well-side (Fig. 10a), in which EDX analysis detected only a high Ca-peak. In the middle portion of the bank, the body consisted of many particles packed closely, where a moderate peak of Ca and a low peak of P were observed. On the tissue-side, the surface was almost covered with solid amorphous lamellae (Fig. 11), where high peaks of Ca and P were detected (Fig. 12). Considerable amount of calcium phosphate-like calcified material was deposited in the chamber. This was found also in 1-week specimens, but the band of high peaks of Ca and P in the 14-week specimens was much wider than that of 1-week specimens. At the edge of the tissue-side, particles were being incorporated into the amorphous lamellated body of the bank as if it were covered with a thin film.

Dystrophic caicification

s. !! Tissue-side of the barrier in Fig. 10b ( x 1000)

Fig. 12. EDX analysis of the solid amorplious lamellae in Fig, 11 (vertical 5000 count).

Histopathological investigation of the biopsied tissue

The connective tissue from 24-h specimens revealed inflammation at the well-side. In 1-week and 14week specimens, very slight or no sign of inflammation was observed. At a contact area on the precipitate-bank, fragments of calcified materials were observed being stained deeply with hematoxylin. No cell could be identified as the specific one ihat promoted calcification or phagocytosis.
Discussion

In previous studies, initial calcification has been related to a von Kossa positive granulation layer beneath a necrotic layer of tissue. However, the role of calcium carbonate-like precipitates produced by neutrahzation of calcium hydroxide with carbon dioxide 11 the tissue, remains controversial. Eda (9) has dem1 onstrated histochemically the deposition of Ca, P and Mg in this layer. He claims that this layer is important m the following calcification, but regards the neutralized precipitates as a matter unrelated to calcifi-

cation. Binnie et al. (5) also has seen deposition of calcium phosphate in the von Kossa positive layer, with no reference to neutrahzed precipitates. Holland et al. (10) find that many large granulations birefringent to polarized light are localized between necrotic and vital pulp tissue, and are accompanied by von Kossa positive granulations around them. They suggest that the neutralized precipitates encourage the pulp to precipitate the von Kossa positive, calcium phosphate granulations. In studies with the transmission electron microscope, Schroder (2) suggests a role for matrix vesicles in the initial calcification, and Kawakami et al. (6) have found needlehke electron dense crystals of calcium phosphate in degenerated collagen fibers or dead cell bodies beneath necrotic tissue. However, they show little evidence how these ultra-fine structures would develop into substantial calcified material. In the present experiments, a sequential process was recognized in the initial tissue reactions, that is formation of a necrotic layer (tissue dissolution) and calcification seen as a rapid precipitation of crystals by neutralization and their prompt growth into a barrier (dystrophic calcification). It was found that additional Ca and P deposited directly on the precipitate particles to combine them producing a riverbank-like structure. Therefore, it is suggested that the precipitate itself had the potential to induce dystrophic calcification from the tissue, which is in agreement with Holland et al. (10). The prcipitatebank had good affinity to microvessels, which seemed important to promote the absorption of Ca and P from the microcirculation. Long lasting leucocyte adherence might arise from high permeability of microvessels needed to supply Ca and P. However, it is not reasonable to regard to precipitates as pure calcium carbonate, because they reacted with vascular tissue. SEM and EDX examination revealed that the precipitate-products of 24hour specimens showed not only a Ca-peak but also weak peaks of P, S and/or Mg at the fused portions between the crystals. P and Mg were usually found at the calcification site (9), whereas S-peaks seemed to indicate that neutralization of calcium hydroxide had occurred by carbon dioxide and organic tissue components. Class et al. (11) report a basophilic layer in the necrotic tissue immediately after calcium hydroxide came in contact with the exposed pulp. They discuss that the layer may consist of calcium proteinate, a product of reaction between calcium hydroxide and tissue protein, and it conceivably might play an important role in the subsequent calcification. In the present concept of calcification, it is known that calcium-protein-polysaccharides as a ground substance have a high afHnity to Ca ions (12). It was assumed that the precipitate product in the chamber was one such calcium com169

Wakabayashi et al.

pound. Mitchell et al. (3) have tested other highly alkaline compounds such as harium hydroxide, and Raferances found that they fail to induce mineralization. Seltzer 1. FOREMAN PC, BARNES IE. A review of calcium hydroxide et al. (13) demonstrate that calcium carbonate and Int Endo 3 1990; 23: 283-297. calcium chloride also fail, and conclude that the 2. SCHRODER U . Effects of calcium hydroxide-containing pulpavailability both of the calcium ion and the hycapping agent on pulp cell migration, proliferation, and differentiation. J Dent Res 1985; 64: 541-548. droxyl ion is needed to induce calcification. In the 3. MrrcHELL DF, SHANKWALKER GB. Osteogenic potential of experiments with the rabbit ear chamber, calcium calcium hydroxide and other materials in soft tissue and carbonate, magnesium hydroxide and barium hybone wounds. J Dent Res 1958; 37: 1157-1163. droxide, all failed to make precipitate-barriers in the 4. RASMUSSEN P, MJOR LA, Calcium hydroxide as an ectopit chamber {unpublished data). The high alkalinity of bone inducer in rats. Scand J Dent Res 1971; 79: 24-30. 5. BiNNiE WH, MITCHELL DF. Induced calcification in the calcium hydroxide is an indispensable factor to subdermal tissues of the rat. J Dent Res 1973; 52: 1087-1091. make precipitates of calcium and organic sub6. KAWAKAMI T , NAKAMURA C , HASEGAWA H , AKAHANE S, stances. In this process, the necrotic zone is left as EDA S. Ultrastructural study of initial calcification in the rat an unavoidable byproduct, and the resulted precipisubcutaneous tissues elicited by a root canal filling material. tates played a leading part in the dystrophic calcifiOral Med Oral Surg Oral Pathol 1987; 63: 360-365^ cation. The high alkalinity of un-neutralized cal7. HOWDEN G F , SILVER IA, MRCVS MA. The use of an improved rabbit ear chamber technique for the study of dental cium hydroxide was soon dammed up from the materials. Int Endo J 1980; J3: 3-16. living tissue by the precipitate-bank and exerted no 8. AHERN JJ, BARCLAY W R , EBERT RH. Modification of the further irritation. rabbit ear chamber technique. Science 1949; W: 655. 9. EDA S. Histochemical analysis on the mechanism of dentine In conclusion, the effect of calcium hydroxide formation in dog's pulp. Bull Tokyo Dent Coll 1961; 2; 59-88, appeared to be that if formed an immediate precipi10. HOLLAND R , PINHERIO C E , MELLO W, NERY MJ, SOUZA V, tate-barrier that induced dystrophic calcification. Histochemical analysis of the dog's dental pulp after pulp When calcium hydroxide is applied to the exposed capping with calcium, barium, and strontium hydroxides. .7 EndodoTi 1982; 8: 444-447. pulp, pulpal cell migration, proliferation and differ11. GLASS RL, ZANDER HA. Pulp healing. J Dent Res 1949; 28: entiation proceed beneath this barrier of dystrophic 97-107. calcification, and new dentine is deposited by the 12. BowNESS J M . Present concepts of the role of ground subodontoblasts (9, 14~17). stance in calcification, Clin Orthop 1968; 59: 233-247. 13. SELTZER S , BENDER IE. Some mfluences affecting repair In apexification (18) and iatrogenic perforation oi the exposed pulps of dog's teeth. J Dent Res 1958; 37: treatment a dystrophic calcification barrier is made 678-687, by calcium hydroxide as well. A hard tissue barrier 14. SciAKY I, PiSANTY S, Localization of calcium placed over is then deposited by odontoblasts or cementoblasts. amputated pulp in dotj's teeth, J Dent Res 1960; 39; These results were obtained in healthy connective 1128-1132, 15. Pis.ANTY S, SciAKY 1, Origin of calcium in the repair wall tissue. In inflamed tissue, a process of destruction after pulp exposure in the dog, J Dent Res 1964; 43: 641 -644, or resorption of precipitate-products might occur 16. YAMAMURA T. Differentiation of pulpal cells and inductive instead of dystrophic calcification. This point should influences of various matrices with reference to puipal wound be investigated by another experiment. healing, J Dent Res 1985; 64: 530-540,
17. SEUX D, COUBE M L , HARTMANN DJ, GANTHIERJB, MAGLOI-

Acknowledgements - The authors wish to thank Dr. Debari in XMA section of Showa University for his great contribution to this study, and L. Stephens, for her valuable advice in preparation of the manuscript.

RE H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxidecontaining cement. Arch Oral Biol 1991; 36: 117-128.
18. MORSE DR, O'LANIC J, YESTLSOY G. Apexification: review of

the literature. Quint Int 1990; 21: 589-598.