Bio-Microscopical Observation of Dystrophic Calcification Induced by Calcium Hydroxide

8/3/2019 Bio-Microscopical Observation of Dystrophic Calcification Induced by Calcium Hydroxide

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Dent Traumatol 1993; V: 165-170in Denmark . At! rights reserved Copyright Munksgaard Endodon tics &Dental Traumatolo

ISSN 0109-2502

io - m ic r o s c o p i c a l o b s e r v a t io n o f d y s t r o p h iclc i f ic a t io n in d u c e d b y c a lc iu m h y d r o x i d e

r bank in the first 48 h. After 1 week, microcircula tion recov-

H a j i m e W a k s b a v a s h i ,M a n a m i H o r i k a w a , A k i y o s h i F u n a t oA t s u s h i O n o d e r a , K o u k i c h i M a t s u mDepartment of Endodontics, Showa UniversitSctiooi ot Dentistry, Tokyo, Japan,

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

um has been dem onstrated by m any clin-

Most of the previous studies were done by histo-

s difficult to know actually the beginning and

nique for i n vivo observation of living vascular tA transparent chamber is inserted into a rabear, and after a thin vascular tissue regeneratethe chamber, a microscopic view of living microculation can be observed continuously in the stissue over weeks and months without anestheThis technique was originated by Sandison andveloped by Clerk & Clerk for the microcircularesearch in 1930'. Then, various modificationchamber design were made and adopted for expmental pathology. In dentistry, Howden et al.apphed this technique to examine microcireulareactions to endodontic and restorative materHowever, calcium hydroxide has not been tesBefore the present experiment, the authors desigan improved rabbit ear chamber to estimate


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a k a b a y a s h i a t a l .um hydroxide in the connective tissue, using the

ion and continuously up to 14 weeks. Then , calci-

) . The basic design followed Ahern's model (8),

One male albino-rabbit (weighing 3.0 kg) wasin an experimental container, and local infil-

Then, one center hole and three small holes aroit were punched on the upper portion of thewith a specially-designed punch. The skin arthe center hole was peeled off, leaving vascularwork on the cartilage as intact as possible. Athat, the chamber disk was inserted, fixed and gto a holder ring with an adhesive agent. The sin the chamber filled with blood, and healing owound progressed. Microvessels were completelgenerated on the observation table usually 5 wafter the operation (Fig. 2).

Eight chambers of 7 rabbits with completegeneration of vascular network were provided.cium hydroxide powder was mixed with salineg/ml, pH 12.5) to a paste consistency used inclinic. The teflon plug was carefully removed aseptic way. Calcium hydroxide paste was appon the tip of the tefion plug, and then the was put back into the well to introduce calchydroxide to the thin vascular tissue on the ovation table. The tip of the removed plug had cut about 0.5 mm short before replacing to mthe volume of the material about 1.5 mm cubeInteraction between the material and the mcirculation inside was observed continuously ua biomicroscope, immediately after the applicaafter 3, 6, 12, 24, 48, 72 h, after 5 days, then, eweek up to 14 weeks. A light microscope equiwith a special observation stage and a camera

used with magnification from 10 to 400.At three different periods of 24 h, 1 week aweeks after the app lication, two chambers each removed under a local anesthesia. The observtables with precipitate products were taken outprepared for SEM and EDX. They were air-dsputter-coated with carbon and examined wiSEM 'JOEL T220A; accelerating voltage: 20 Then, ultimate analysis of the precipitate prod

Design of the improved rabb it ear cham ber (length: mm]c: observation table, d: well. A


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Dystrophic calcification by Ca|

nts with an ED X (Kevek DELTA IV ; acceler-

at the same time, fixed

From 6 to 48 h, the precipitates were increasing

:")). After 72 h, the rapid formation of precipitate-

mens from 100 to 200 ^m. In side

One week after the application, microcirculation

bank, newly-formed capillaries were growing ithe precipitates (Fig. 6). No pathological featuwere noticed in their form and function, excslight leucocyte adherence remaining up to 7 weFrom 2 to 14 weeks, the findings on the obvation table were constant without any specchanges to be mentioned. The precipitate-bankvealed good compatibility with the connective tisand microcirculation, being stable without disingration or resorption. The edge of the bank on tissue-side appeared to change gradually intoamorphous and smooth form, while on the well-of the bank the precipitates remained particle-in form (Fig. 7).SEM and EDX examinationIt was found that all the precipitate productsthe chamber firmly adhered to the surface of observation table. SEM observation of 24-h spmens revealed that the precipitates were in form

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


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a k a b a y a s h i e t a i .

Observation table 7 weeks after the application. A: M icro-aste) , *: central stop. Fig. 9. SEM findings of the precip itate-prod ucts 'i4- h alteapplic ation ( x 150), A: tissue-side, B: well-side.

SEM tindiii:licat ion ( x ir>Oj

c:s o\ ' the obser\'atinn table 24 li aftera: (altium t.arbonate-like precipitate; b:

well (calcium hydroxide paste). ? ; 1( 1 S L M t i i i d u i ^ ' s o tV lUOi A t ibs ue-s id t ,

r > ' s t a b .j .nni I M W H ks alt( i the app iuuell-side, a btc-hn e-shap ed

rystals of calcium carbonate (Fig. 8). This crystalhese crystals fused to larger clusters here and theren the observation table around the well. Amor-phous deposits between fused precipitates showed aower Ca-peak and also weak peaks of S and P.In 1-week speciments, the precipitated bank wasver 200 |im wide. Fusion of the crystals advancedmore on the tissue-side of the bank (Fig. 9). Onlya high Ca-peak was detected from single crystals atthe well-side, whereas a medium Ca-peak and a lowP-peak were detected in the middle portion of thebank. In some areas, weak peaks of S and Mg were

also found. On the tissue-side, both high Ca- andhigh P-peaks similar to calcium phosphate were

of the bank were different from portion to porOn the well-side of the bank, precipitates beconsiderably larger in size, but less in nu mber.hive shaped large crystals about 100 |im in diamwere found on the well-side (Fig. 10a), in wEDX analysis detected only a high Ca-peak. Imiddle portion of the bank, the body consistemany particles packed closely, where a modpeak of Ca and a low peak of P were observedthe tissue-side, the surface was almost covered solid amorphous lamellae (Fig. 11), where peaks of Ca and P were detected (Fig. 12). siderable amount of calcium phosphate-like calcmaterial was deposited in the chamber. Thisfound also in 1-week specimens, but the banhigh peaks of Ca and P in the 14-week speci


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Dystrophic caicification

!! Tissue-side of the ba rrie r in F ig. 10b ( x 1000)

EDX analysis of the solid amorplious lamellae in Fig,

1-week and 14-

l could be identified as the specific one

cium carbonate-like precipitates produced by neu-hzation of calcium hydroxide with carbon dioxide

cation. Binnie et al. (5) also has seen depositiocalcium phosphate in the von Kossa positive lawith no reference to neutrahzed precipitates. land et al. (10) find that many large granulationrefringent to polarized light are localized betweencrotic and vital pulp tissue, and are accom panievon Kossa positive granulations around them. Tsuggest that the neutralized precipitates encouthe pulp to precipitate the von Kossa positive, cium phosphate granulations. In studies withtransmission electron microscope, Schroder (2) gests a role for matrix vesicles in the initial calcation, and Kawakami et al. (6) have found neehke electron dense crystals of calcium phosphadegenerated collagen fibers or dead cell bodiesneath necrotic tissue. However, they show littledence how these ultra-fine structures would devinto substantial calcified material.

In the present experiments, a sequential prowas recognized in the initial tissue reactions, thformation of a necrotic layer (tissue dissolution)calcification seen as a rapid precipitation of cryby neutralization and their prompt growth inbarrier (dystrophic calcification). It was foundadditional Ca and P deposited directly on the cipitate particles to combine them producing aerbank-like structure. Therefore, it is suggestedthe precipitate itself had the potential to indystrophic calcification from the tissue, which agreement with Holland et al. (10). The prcipibank had good affinity to microvessels, wseemed important to promote the absorption oand P from the microcirculation. Long lasting lecyte adherence might arise from high permeabof microvessels needed to supply Ca and P.However, it is not reasonable to regard to pretates as pure calcium carbonate, because theyacted with vascular tissue. SEM and EDX exaation revealed that the precip itate-pro duc ts of

hour specimens showed not only a Ca-peak but weak peaks of P, S and/or Mg at the fused porbetween the crystals. P and Mg were usually foat the calcification site (9), whereas S-peaks seeto indicate that neutralization of calcium hydrohad occurred by carbon dioxide and organic ticomponents. Class et al. (11) report a basoplayer in the necrotic tissue immediately after cium hydroxide came in contact with the expopulp. They discuss that the layer may consiscalcium proteinate, a product of reaction betwcalcium hydroxide and tissue protein, and it ceivably might play an important role in the


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W a k a b a y a s h i et al.pound. Mitchell et al. (3) have tested other highlyalkaline compounds such as harium hydroxide, andfound that they fail to induce mineralization. Seltzeret al. (13) demonstrate that calcium carbonate andcalcium chloride also fail, andconclude that theavailability both of the calcium ion and the hy-droxyl ion is needed to induce calcification. In theexperiments with the rabbit ear chamber, calciumcarbonate, magnesium hydroxide and barium hy-droxide , all failed to make precipitate-barriers in thechamber {unpublished data). The high alkalinity ofcalcium hydroxide is an indispensable factor tomake precipitates of calcium and organic sub-stances. In this process, the necrotic zone is left asan unavoidable byprodu ct, and the resulted precipi-tates played a leading part in the dystrophic calcifi-cation. The high alkalinity of un-neutralized cal-cium hydroxide was soon dammed up from theliving tissue by the precipitate-bank and exerted nofurther irritation.

In conclusion, the effect of calcium hydroxideappeared to be that if formed an immediate precipi-tate-barrier that induced dystrophic calcification.When calcium hydroxide is applied to the exposedpulp, pulpal cell migration, proliferation and differ-entiation proceed beneath this barrier of dystrophiccalcification, and new dentine is deposited by theodontoblasts (9, 14~17).In apexification (18) and iatrogenic perforationtreatment a dystrophic calcification barrier is madeby calcium hydroxide as well. A hard tissue barrieris then deposited by odontoblasts or cementoblasts.These results were obtained in healthy connectivetissue. In inflamed tissue, a process of destructionof precipitate-products might occurof dystrophic calcification. This po int should

- The au thors wish to thank Dr. De-bari in XMA section of Showa University for hist contribu tion to this study, and L . Stephens, forn preparation of the m anuscript.

R a f e r a n c e s1. FOREMAN PC , BARNES IE . A review of calcium hyIn t Endo 3 1990; 23 : 283-297.2. SCHRODER U . Effects of calcium h ydroxid e-conta inincapping agent on pulp cell migration, proliferatiodifferentiation. J Dent Res 1985; 64 : 541-548.3. MrrcHELL DF, SHANKWALKER GB. Osteogenic pote

calcium hydroxide and other materials in soft tisbone wounds. J Dent Res 1958; 37 : 1157-1163.4. RASMUSSEN P , MJOR LA, Calcium hydroxide as anbone inducer inrats. Scand J Dent Res 1971; 79 : 245. BiNNiE W H , MITCHELL DF. Induced calcificationsubdermal tissues of the rat. J Dent Res 1973; 52 : 1086. KAWAKAMI T, NAKAMURA C, HASEGAWA H, AKAH

EDA S. Ultra struc tura l study of initial calcification insubcutaneous tissues elicited by a root canal filling mOral Med Oral Surg Oral Pathol 1987; 63 : 360-365^7. HOWDEN GF, SILVER IA, M R C V S MA. The use ofproved rabbit ear chamber technique for the study omaterials. In t Endo J 1980; J3 : 3-16.8. A H E R N JJ, BARCLAY WR , E B E R T RH. Modificationrabbit ear chamber technique. Science 1949; W: 659. EDA S. Histochemical analysis on the mechanism of formation indog's pulp. Bull Tokyo Dent Coll 1961;

10. HOLLAND R, P IN H E R I O C E , M E L L O W, N E R Y MJ, SOHistochemical analysis of the dog's dental pulp aftcapping with calcium, barium, andstrontium hydr.7 EndodoTi 1982; 8: 444-447.11. GLASS R L , ZANDER HA. Pulp healing. J Dent Res 197-107.12. BowNESS J M . Present conce pts of the role of grounstance incalcification, Clin Orthop 1968; 59 : 233-2413. SELTZER S, BENDER IE. Some mfluences affecting

oi the exposed pulps of dog's teeth. J Dent Res 19678-687,14. SciAKY I, PiSANTY S, Localization of calcium placeamputated pulp in dotj's teeth, J Dent Res 191128-1132,15. Pis.ANTY S, SciAKY 1, Origin of calcium in the repaafter pulp exposure in the dog, J Dent Res 1964; 43 : 616. YAMAMURA T. Differentiation of pulpal cells and ininfluences of various matrices with reference to puipalhealing, J Dent Res 1985; 64 : 530-540,17. SEUX D, COUBE M L , HARTMANN DJ, G A N T H I E R J B , MRE H. O dontoblast-like cytodifferentiation of hum anpulp cells invitro in the presence of a calcium hyd

containing cement. Arch Oral Biol 1991; 36 : 117-1218. MOR SE DR, O ' L A N I C J, YESTLSOY G. Apexification: rethe literature. Quint Int 1990; 21 : 589-598.


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Bio-Microscopical Observation of Dystrophic Calcification Induced by Calcium Hydroxide