EXPERIMENTAL PULPOTOMIES IN RAT MOLARSDAVID S. BERMAN, B.D.S. L.D.S.R.C.S., M.S., AND MAURY MASSLER, D.D.S., M.S.

University of Illinois, College of Dentistry, Chicago, Ill.

THE purpose of this investigation was (1) to develop further a technic forperforming pulpotomies on rat molars, (2) to study the pattern of pulpal

healing following amputation, and (3) to compare the effects of calcium hy-droxide and of zinc oxide and eugenol on the pattern of pulpal healing in themolars of rats.

It has long been realized that pulpal amputation must be carried out andstudied on a large scale under precisely controlled laboratory conditions inorder to evaluate more accurately the process of pulpal healing. For thispurpose, the rat is a suitable animal because it is easily available in largenumbers and is comparatively cheap. However, because of the small size ofthe rat's teeth, it first becomes necessary to evolve a reliable method of per-forming pulp amputation and pulp capping in the rat molar. Furthermore,this technic should be easily reproduced by other investigators in this field.

Mohammed and Schourl Maurice and Schour,2 Perreault, Massler, andSchour,3 and Silberkweit, Massler, Schour, and Weinmann4 have evolvedsimple methods for operating on the incisor and molar teeth of rats in orderto study the pulpal reaction to various filling materials. Massler, Perreault,and Schour5 and O'Malley6 adapted the above methods to the study of theeffects of pulpal amputations in the continuously growing incisor of the rat.

Miyamoto7 successfully performed surgical amputations on the pulps ofrat molars. His investigation has made it possible to develop further a technicfor performing pulpotomies and to study many of the features of pulpal heal-ing in a molariform tooth of limited growth.

METHODS AND MATERIALS

Pulp amputations were performed in 122 upper first molars of 61 malehooded rats. Forty-five animals were 60 to 90 days of age, while 9 were 30days old, and 7 were 220 or 360 days of age. The 2 latter groups were partof a pilot study on the effects of age on the rate of pulpal healing.

The animal was anesthetized by an intraperitoneal injection of Pentothalsodium, 1 per cent, and placed on an operating table on its back. The tongueand cheeks were reflected from the field of operation. Instruments and burswere modified to suit the size of the tooth as described by Maurice and Schour.2Aseptic conditions were attempted but not always achieved.

An attempt was made to remove dentin splinters with an air spray, butthe histologic sections showed this to have been unsuccessful in a number ofcases. If there was marked hemorrhage, a paper point was used to remove

Received for publication July 6, 1957; revised by authors Jan. 8, 1958.

229

2634)0 iIE'ltRIAN AND1IMASSLICJR, J. I), Rcs.Api-lI, 1958

the excess whole blood, but no deliberate attempt wals maidlel ait the tune ofthe operation to influence the bleeding time or the character of the clot. Sornieexposures were nonblceding or had very little l)leed(ing. The presence of abloodl elot and its character were filllly deteriviined histologiclally.

Fig. 1. Photomricrograph showing pulpal healing 28 (lays after surgical amputation andcovering with calcium hydroxide (Henaitoxylin and eosin; Orig. mag. X300). Note character-istic zones. M, medicament; I, zone of pulpal degeneration; C, primary calcific bridge;D, permanent dentin bridge; 0, odontoblastic lay er.

Two mnain operative approaches were investigated, oe from the, mesialsurface of the first iiolar and the other from the ocelusal surface. It was

found that the ocelusal *approach via the middl-e pulp horn was SuperiOr tothe mesial approach for producing an exposure which eollll le controlled interms of size and depth. Only half of the 28 fillings placed in the niesialsurface were retained while 89 per cent of the 94 fillings placed in the ocelusalsurface were retained, even after 28 days.

The medicamnents used were ealciumt hydroxide and zinc oxide and ent-genol. Each medicament was l-laced in 61 teeth, alternating on right andleft side in successive animals. Each was placed in direct contact with thepulp tissue whxrlever possible. When calcium hydroxide was used, the powderwas dusted into the cavity, covered. with para(ffin wax and then sealed withamnalgamii. The zinc oxide, anmd euigenol cappitigs were also sealed with amal-gailii.

Approximiiately equal numbers of animals were sacrificed at intervals of7, 14, 21, and 28 days following the operation. The animals were decapitatedand the jaws fixed in 10 per cent formalin. The specimens were decalcified,

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IEX1T F I 1MENTAALTPULPOTOM I ES

embedded in paraffin or celloidin, and sectioned at 1-0 microns. They werestained with heiiattoxylin a1nrud cosin or with Mallor-y's triple conneetive tissuestaiin.

Fig. 2. Phiotomicrograph showing pulpal reaction to zinc oxide and eugenol 7 (lays aftersurgical amputation (Hematoxylin and eosin; Orig. nmag, X80). Note zinc oxide and eugenol(1/) in direct contract with pulp tissue -ind increased vascularization.

YI1N I)IN (GS

Fattern andl(hronology of Ilealiny.-The most important finding was the fact that healing followed a definite

pattern under large or small exposures and under both types of medicamnentused. Healing took})lace by the formation of different reaction zones belowthe amputation site. rrhese were seen clearly in specimens obtained at 21 to28 days. Fig. 1 shows the characteristic zones: the layer of mitedicanicut, thezone of pulpal injury and (legeneration, the primary ealcific bridge, the pernia-nent bridge of repa native dentinh, the odontoblastic layer, and normal pulptissue.

231

232 B-E.RMTAN AND ATASSLER J. D. Res.April, 1958

M((/icaml(tt lf(ti: Tlhe zole iil)0X't the arie 1o amiipiitatiii w8s usuallyCOeoiijOd Of0lrei'liinanlsX of Ilieiedieaimeuit either alo-e oi iiiixed with 10-)(ood o(let1ris. Zine oxlde 11 ('1engeiiol tdeil(dl to rei'aiil above the layer1 oif wholeblood and (litd iiot mix with it, wlxve (alciiiiii hydroxide wias usually mixedwith 1l)1o(1 fand causeid1(11 its lieiiiolysis (FPigs. 8.2 and 6).

Fig. 3.-Photoinicrograph showing pulpal reaction to calcium hydioxide 7 (lays aftersurgical amputation (Hematoxylin and eosin; Orig. mag. X80). Note that medicament (11) isadmixed with heniolyzed blood. 1, indicated coagulated pulp tissue. Zone of degeneration (I)is proiinent and there is seen beginning of formation of primary caleific bridge at C'.

When a layer of bloodI (-)1 tissue exudate was foundbItween th iiediea-itierit all(1 the pulp tissue, this te(lnedd to mdlitxfy'and oliliite the action of theIiietdiea1.iiIent.

Zovu Of P1)011)l inl/107jur11j(1artmgenerations: The superficial portion oV theaiti utaptedPll p was usually Iiiaeerateol, mIe('rotie or, eoagulcted. p1'ht'depthInd der('ee injury inl this aIeaI leleied oio the amount of traiitiia ean)sedby the-i operation. anl1d the eg ailatit-ing effect of the Ilidiealornent (I-ppluto1. A

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EXPEIRI M ENT'Al,rU.ITTOM'(TE8()MES 233

thick lawyer () eomagulatdel )ulp tissue dlvelol e unler calcium hlydroxide,while under zinc o)xidle ati cugetiol little ()1w lit) neiesis (o thle superficiallayer ()f alil)litatt(l JIli)tipsse wa alt)lxsrllt (Iigs. 2 and 8).

The (leple)G lportion oh this zmici was clriieterized(l ) (deelegnratixve chanlges.l'he( intetreellular matrix (pink-staining appeared to' )e 1110mCe p1roiiiiiienit andeniclosed degcllcrtilig eolls (Fig. 4). rThis layer wais first s(een at 7 (lays andbccaiiie more promiiiiiet onI longer survival.

Primary (ct/rific bridge: At approxim-itely 14 days, bel()w the zonte of in-jluy antid tlgetieziatiotii there wa.s seen a lblnc-sttailliiig layer o)f (dyStio()phicc(al(ifiCationII. It Wxxas char1Itacterized by the lippear-afl'ite of large tel't'gaiteatiligl)ull)al cells etal')pe(d withill (ltdllse network oh1fibrot)us stlands (Figs. I1, 4

Fig. 4. Photornicrograph showing pulpal reaction to zinc oxide and eugenol 21 days aftersurgical amputation (Hernitoxylin Hand eosin; Orig niag. X130). Note the characteristic zon-ing. 1, zone of degeneration; C, primary calcific bridge. Permanent dentin bridge (D) show sw ell-organized, tubular arri angement.

ntI 5). These fiber_s (orI thiel enve olong eminenting substances) staitied a

deep blue coltOr with henitiatoxylin, leading to the conclusion that thley wveeundeIrgoing calcification. The tellL dystrlohi( refes to the fact that the en-trappedi cells were in a state of degeneration.

The zonle of d1ystrophic calcification <always appeared 1)etwxeen the zone ofpullpal degeneration al)ove and the normal 1)pul) tissue below. It thus formeda pr.--otective ealeifie br-idge over the vital pulp tissue and ])recede(l the ap-pearance of the true dientin l)riilge forimiedlaCter lby odontoblastic (s(118. Forthis reason it is referredl toi) as the `primaliy ealtifie. lbridge." Lan thickness, it,lrallged fPromo a thin line (Fig 1) to a thick, well-differentiated ztone (Fig. 4).

234 BElRMA.N AND) MASSLEAR J. D. Res.April, 1958

Its size appea-rted to depend on tfhe natue( of t-he zonealt )O\e. Whelon the zonle.of pulpl-a. inl jiy11abhove shlowX\ed a. slow progressive caltd11(10iid (leellerl'-iln, thlepiltraliry ealeifiet bhnidge forlle(l slowly a-ind late, wa(1s nlot well orgatn1lized allndtended to be wide. When thflie zone alomve showed a1 1aIlpil, co(a-gla1itilng nelerosis,the primcar-y ealecific)irie appe el a rI, was dense el l lgized andnarrow. [n the latter instanee, Tormition of the pei'anment dleiitiil hridg'e)egattLllealy, soonaIfter the aippearancile of the priiayalevctifie bridge.

Fig. 5. Plhotomicrograph. show-ing wide primary calcifie bridge under calcium hydroxide 28(lays after surgical amputation (Hematoxylin and eosin; Orig. iag. X3300). Note large d(e-generating cells entrapped within calcified fibrous strands. I, zone of degeneration.

The presence of a primary calcific bridge was the most constant andcharacteristic feature at -14- (laiys after pulpal amputation. It appeared to actas an organizer for the cells involved in the dentin formation and bridgingthat followed. It obviously represented an early walling off of the injuredand degenerating portion of the pulp from, the vital pulp tissue below.

Permanent bridge of reparative dentin: At 21 days, it was possible todetermine whether complete pulpal healing would or would not take place.Those specimlens, in which a thin primary bridge had formed, usually showed

Volume 37 EXPERIMENTAL PULPOTOMIES 235Number 2

a well-developed layer of true dentin bridging the site of amputation. Speci-mens which showed a wide, irregular primary calcific bridge were much slowerin stimulating the formation of a dentin bridge. In such cases, the amountof secondary dentin formed at 21 days was much smaller (compare Figs. 4and 5).

That portion of the permanent dentin bridge which was formed first, incontact with the zone of dystrophic calcification, tended to be irregular instructure (Fig. 4). The portions formed later were more regular.

The dentin bridge extending across the wound site was made up of 2components. The central portion was formed by newly differentiated cellsand therefore tended to be irregular in structure. The lateral portions wereformed by old odontoblasts lining the wall of the dentin adjacent to the siteof amputation. These cells seemed to be stimulated to renewed activity bythe amputation and formed a thick layer of regular, tubular dentin which,under a small exposure, almost bridged the entire gap (Figs. 1 and 4).

Vital Pulp Reaction.-Vascular reaction: A very prominent feature of the healing pattern was

the increased vascularization of the pulp tissue immediately below the zonesof reaction. It was very prominent at 7 days and less prominent, but stillpresent, at 14, 21, and even at 28 days.

At 7 days after amputation, there was a marked increase in the numberof capillaries in the pulp just below the zone of injury (Figs. 2 and 3). Thesevessels were always dilated and at times ruptured causing small hemorrhagesinto the pulp tissue.

The blood vessels seemed to retreat as the primary calcifie bridge and thepermanent dentin bridge were formed and increased in thickness. Finally,these vessels formed a subodontoblastic plexus below the cells that formed thepermanent bridge. The increased peripheral vascularization may be a reac-tion of the vital pulp to the trauma of the operation but, since it is presenteven in later specimens, it is more likely to be an integral part of the healingprocess.

Cellular reaction: The odontoblasts at the site of amputation were se-verely injured and consequently degenerated. However, at a slight distancefrom the site of amputation, the odontoblasts were strongly stimulated andproduced large amounts of secondary dentin rapidly, forming the lateral mar-gins of the dentin bridge (Figs. 2, 3, and 4). At a farther distance, the odonto-blasts appeared unaffected by the operation.

The primary calcific bridge was probably the product of degeneratingcells whereas the permanent dentin bridge was formed as a result of vitalcellular processes. It appeared that approximately 14 days were required fornew odontoblasts to differentiate and at least 21 to 28 days before they pro-duced a regular type of tubular dentin which formed the main portion of thepermanent dentin bridge.

Comparison Between Calcium Hydroxide and Zinc Oxide and Eugenol.-The basic pattern of pulpal healing described above was observed under

both calcium hydroxide and under zinc oxide and eugenol. Minor differences

W2:3(; iKR.M \N \NI) I \ S A J 1. I). Res.A.rill 1958

were evX;idenit at 7T aId 14 dlays. Calcium hydroxide had{l a distinct inecrotizingor coagulating effect on the superficial portion of the amputated pulp and onany inter ending 0loo0( or exudaIte. IThis appeared to because aIn early alppear-aiicc (often at 7 da)iys) of the zone orf dystrophlie caflcificaition (Fig. 3).

Whien theie zinc oxide and etigenol Aere in directt contact With tle p)111), thlettriderlyuig tissue aippecaired to be norlud oitor conta-iined a ha rge n uml)er ofoIvnlrl)holtllc.le' i~leukoeytes (Vig. 6). When Potynlorlholimlear Ilcukocytes

jX r; QW>4, 5; t>+56~~~~,V 4

Fig. 6.-:Photomicrogralphl showing an infiltration of polyniorphonuclear leukocytes andred blood cells xs ithin the pulp under zinc oxide and eugenol 7 (day s after surgical amputation(fl-ematoxylin and cosin; Orig mag. X80> )

welI (olbse I 1m ud11dert zintc. od,Icanti eugenoll t1hey appeared to hse aissoc..iatetilwith an infiltrlation ol red 1)10(11 (clils into the tissue at 7 tla-ys aftr aill)litation (Fig. 6). By 14 daI-ys, the muinbthr of pdlyniorphonuclean' len)ocytes wa-t-sgrefatly reduced and at 2 L deays, they were no longer evident.

At 14 (daiys it beeanie eeanr that the basie plattern- of healing was pro-gressing ini a par.-allel fashion below both unedicaments. Specimens covered

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EXPTEflMIENT-A.T1 UIlJ ,POTOMATIIfES 237

with eatleinm hlydlr-oxidle shoved( wa ell-orineti p1rinlary c'lc1ific bridlge. Solmespeenimens were well adraneeri i the formiaition of the pernrane,1nt dentinbridge. The 1)atten- of heal,1ing ill pulplIs eoveled with zilnecoxidele cand eugenolappeared to be slightly delIyed. At 1.4 (days the p:)r-imarty e(aleific bridge wasonly beginning to ["ma. The pulp tissue betweenl the primaru-y ealcifie bridgea-lnd the niedicani-ent now showed evilenlle of1 legenertiioll with all increasedprotminence oJf the intereellular i atriix.

By 21 days the attendn of pnlpal heallinig unIeir 1)th itedicaroents wasapproximately the sa!rtme. IUndercle(-ininal hydroxide, the peliianent dentinbridge was well estaablishedl. Tinder zinc oxide and eugenol., the dentin br-idgewas also well formed. Apparienttly, the cells under the zine oxide and eutgenolhad eltaught ul) with those unler calcium hydroxi\de anl the histologic picturewas the sa-me in 1oth groups of specimeins (Vigs. 1 anid 4).

Fig. 7.-Photomicrogra Eph showing a delayed pulpal reaction 7 (lays after surgical ampu-tation and covering with zinc oxide andl euLgenol (Hemiatoxylin and Cosin; Orig. mag. X80).Note large pool of whole blood overlying amputatedl pulp surface. This has retarded rate ofpulpal healing. There is no coagulation of tissue nor zone of degeneration yet eixdent. J, areaof niedicament lost during sectioning.

At 28 days, it was- (lifficult to distinguish between amlllttte(1 pulps treatedwith ealeinni hydroxide or Zili)e oxi(le a-tnd engeliol. rlThe expose n111der eaCCchediedcament watis scaled off' ly a newly formlled dentin bridge. 'The lplps were

normal and there was no evidence of chrioic. inflalaunaltion. lliler the calciumhydroxide or the zine oxide aidi eugernol.

Although sonie differences (lid exist at 7 and -14 (lays between specimensecoTer1'ed with eale(iuii hydroxide or zince oxide al(1 thgenl,these differences

g28 BERMAN ANT) MASSLER TI. D. Res.April, 1938

were no longer ap)p(arient ait 28 dlays. This suggests that the pulp has a patternof healing which caii be acceler1a1ted Orl retfIalWed to somie (legree lbllt whichcannot be a]terledtl basiea.lly by tih mle-lia-lllclit used.

Effects of Blood on I) alJal IHallnig.In many specimens at br-oad layer of blood (whole or hemolyzed) or exu-

date intervened between the mclie(hlllent and the pulp. In these specimens,the pulpal reaction wa-ris obviously related to this covering rather than to theiiediecaiuent.

Fig. 8.- Photomicrograph showing the effect of the loss of filling material (Hematoxylinand eosin; Orig. mag. X80). Note the 3 zones of diffuse dystrophic calcifications (0). Alter-nating zones probably indicate successive attempts by pulp to seal off exposed area but con-tamination has resulted in pulpal degeneration.

When a layer of whole blood (not coagulated or clotted) covered the am-putated pulp, the effect was to retard the degeneration of the underlying tis-sue and thus also to delay the appearance of the primiiary and permanent den-tin bridge. This appeared moil-e often under zinc oxide and eugenol (Fig. 7).

Under calcium hydroxide, when a layer of blood intervened between theiiiedieanient and the pulp, the blood cells were coagulated (Fig. 3). In suchspecimens, the rate of healing cappealr ed to be somewhat accelerated.

In no ease was clotting or organization of the blood observed. Whenblood was present over the pulp, it relnained throughout as a pool of wholeor hemolyzed but unorganized blood, depending on the medicament used. Thereason for the failure of the blood to clot and become organized is not clear.

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EXPERIMENTAL PULPOTOMIES

It was clear, however, that the presence of such a pool of blood generallyslowed down the pattern of healing probably by interfering with the actionof the medicament on the pulp itself. This was seen more often under zincoxide and eugenol than under calcium hydroxide since the latter tended tocoagulate the blood and thus to accelerate the rate of healing.

Effect of Loss of Fillings.-In specimens from which the fillings were lost or when marginal leakage

had occurred, healing did not take place. In these, extensive pulpal degenera-tion invariably followed.

It was possible to determine approximately when the filling had been lostby measuring the amount of secondary dentin formed along the dentin wallsadjacent to the site of amputation and by observing to what stage the patternof healing had progressed. Many pulps, although exposed to oral fluids, stillattempted to seal off the exposure by the formation of a wide zone of dys-trophic calcification (Fig. 8). This demonstrated the great recuperativepowers of the pulp and emphasized (to us) the important fact that the successor failure of bridging under a pulpotomy depends more on the integrity ofthe filling and the adequacy of the marginal seal, than upon the substanceused to cover the amputation.

DISCUSSION

Stages in Pulpal Healing.-This study emphasizes the fact that each stagein the healing process is dependent upon the presence and character of thepreceding morphologic zone (Fig. 9). It was seen that calcium hydroxidecoagulates and necrotizes the superficial pulp tissue as described by Glassand Zander.9 This appeared to stimulate the rapid formation of the primarycalcific bridge. On the other hand, zinc oxide and eugenol did not causenecrosis of the superficial pulp tissue and degenerative changes were delayed.This resulted in a comparatively late formation of the primary and the perma-nent dentin bridges.

The basic reaction observed during pulpal healing was a protective wall-ing off of the site of injury by a fibrous calcification of the peripheral portionof the degenerating pulp at its junction with the vital pulp tissue (the primaryealcific bridge). This temporary bridge allowed the vital portion of the pulpto organize its cellular elements toward the formation of a more permanentseal, the permanent dentin bridge.

The formation of a primary calcific bridge represents the primary de-fensive reaction of the degenerating but still vital pulp cells. O'Malley6 re-ferred to this as an area of dystrophic calcification while Fish8 called it a truecalcific degeneration. It is interesting to speculate as to the source of thefibers and the calcium salts necessary for the calcification of this zone. Arethe fibers a product of the degenerating cells (fibroblasts?) entrapped in thearea or are they precipitated or polymerized from the protein matrix in thearea? Do the calcium ions come from the underlying vital pulp or from the

239

240 BERMAN AND MASSLER J. D. Res.April, 1958

medicament above? Or are the calcium salts contained within the fibers andcells in the area, being precipitated during the process of "calcific degenera-tion"? This study indicates that the calcium is not derived from the medica-ment above, else the primary calcific bridge would not be formed under zincoxide and eugenol.

The primary calcific bridge may be regarded as the organizer of the heal-ing process in that it is always seen prior to the formation of the permanentdentin bridge. It has been postulated that a calcified material must be presentto provide a stimulus for the differentiation of new dentin-forming cells. Itwas observed that a thin zone of dystrophic calcification leads to the earlyformation of a thick permanent bridge while a wide diffuse primary bridgeleads to a thin and late permanent bridge. This relationship was also notedby Miyamoto.7

PROGRESSIVE STAGES IN PULPAL HEALING

ZOE Blood INJURY (Amputation) Ca (OH)2Sedates InuTes ma(I day)

|INFLAMMATION

Slows down H (7 days) CoagulatesReaction r-DEGENERATION 4

I| AcceleratesFIBROTIC REACTION Reaction

+ Zone of dystrophic

CALCIFICATION 1..calcification

( 14 days)PlPulp

*t s tDegeneration

VASCULARIZATION fVital pulpa reaction

DIFFERENTIATION OF CELLS

| (21 days)

IRREGULAR REPARATIVE DENTIN

I (28 days)

IREGULAR DENTIN FORMATIONFig. 9.-Diagram showing progressive stages in pulpal healing.

The Permanent Dentin Bridge.-The term "permanent dentin bridge" isused here to describe a dentinlike material formed by vital cells (odontoblastsor odontoblast-like cells) which reaches from one lateral margin of the ex-posure to the other. A question that has often been raised is the source ofthe cells that form the permanent dentin bridge. The findings in this investi-gation suggest that both old cells, which have migrated from the dentinal walls,and new cells enter into the formation of the dentin bridge. It was observed that

Volume 37 EXPERIMENTAL PULPOTOMIES 241Number 2

the quickly formed dentin bridges under very small exposures were laid downprimarily by surviving odontoblasts from the lateral walls near the exposure.Under large exposures, the main portion of the bridge was formed by newlydifferentiated cells aided by old odontoblasts lining the lateral dentin walls.

Effect of Medicament.-The findings in this investigation indicate thathealing of surgically amputated pulps takes place below zinc oxide and eu-genol as well as under calcium hydroxide. The pattern of healing, as de-scribed in this paper, does not support the thesis that an exogenous calciumsalt is essential to pulpal healing or bridging of the amputation site. Themost important evidence is the fact that (a) bridging can and does take placeunder nonealcium materials in direct contact with the pulp, and (b) calcifica-tion of the primary bridge takes place at an area farthest from the medica-ment and at the junction of the vital pulp tissue. All the evidence points tothe vital pulp tissue as the "reactive" zone and the source of the calcium ionsnecessary for calcification of both the primary and permanent bridges.

Minor differences were observed between the pulpal reactions under cal-cium hydroxide and under zinc oxide and eugenol. These differences wereconfined entirely to the first 14 days and were related to the rate of healing-not the type or quality of healing. It is clear that the differences were theresult of the effect of these substances upon the superficial layer of the pulponly (Fig. 9). Once the superficial zone had degenerated (after 14 days) andthe medicament was separated from the underlying vital pulp tissue by a widezone of degenerated and calcified material, the influence of the medicamentdisappeared or at least became much less apparent.

Calcium hydroxide had a necrotizing effect on the contiguous pulp tissueor blood. Characteristically, the pulpal reaction below this coagulum pro-ceeded more rapidly than under the relatively less injured tissue covered byzinc oxide and eugenol. These observations with reference to calcium hy-droxide have been reported previously.9

This study showed that many (but not all) of the pulps capped with zincoxide and eugenol had an aggregation of polymorphonuclear leukocytes in thesuperficial pulp tissue at 7 and 14 days after amputation. This confirms thefindings of Glass and Zander9 and O'Malley.9 However, pulps examined 21and 28 days following amputation did not show a persistence of these in-flammatory cells.

Loss of Filling.-This study re-emphasized the common clinical observa-tion that pulpal healing will not take place if the filling is lost or leakage oc-curs. This factor, more than any other investigated in this study, determinedwhether or not the pulp would heal.

SUMMARY AND CONCLUSIONS

Surgical amputations were performed on the pulps of 122 maxillary firstmolars of 61 albino rats. Half of the amputated pulps were covered with cal-cium hydroxide and half with a zinc oxide and eugenol paste. The animalswere sacrificed at 7, 14, 21, or 28 days and the teeth subjected to histologicanalysis for the purpose of studying the pattern of pulpal healing.

242 BERMAN AND MASSLER J. D. Res.April, 1958

It is believed that a suitable technic has been developed for performingpulpotomies on the first maxillary molars of the rat. The method is repro-ducible and may be used for further investigations into the effects of age andvarious medicaments upon pulpal healing following surgical amputation. Forthese studies, the rat molar is a suitable biologic test object.

1. In the rat molar a stereotyped pattern of healing was observed aftersurgical amputation of the pulp. This pattern was basically the same underany of the medicaments used.7 It was characterized by the formation of asuperficial zone of pulpal injury and degeneration under which a layer ofdystrophic calcification was formed followed by the building of a true dentinbridge. The primary calcific bridge appeared to be directly related to, andessential for, the differentiation of new odontoblasts and the formation of atrue dentin bridge. The outstanding characteristic of the healing pattern wasthat the formation of each zone was dependent upon the presence and char-acter of the layer above (Fig. 9).

2. Although differences in the pulpal reactions under calcium hydroxideand zinc oxide and eugenol could be observed at 7 days after amputation, by21 days the histologic picture was approximately the same. Calcium hy-droxide is therefore not essential to pulpal healing and bridging. However,calcium hydroxide caused a more rapid necrosis and degeneration of the super-ficial layer of pulp tissue and a more rapid appearance of the primary calcificbridge. The action of the calcium hydroxide in promoting healing may be onthe basis of its high alkalinity and coagulating action rather than on its cal-cium content since healing also occurred under zinc oxide and eugenol albeitat a slower rate.

3. Unorganized blood seemed to retard healing while precipitated or co-agulated protein material (pulp tissue or exudate) appeared to favor pulpalhealing.

4. A prime requisite for pulpal healing appeared to be an adequate sealagainst the ingress of oral fluids. This factor overshadowed all others. Inthis study, the medicament used did not appear to be the major factor in pro-moting the bridging of the exposure.

The authors wish to thank W. M. Winn for making the photomicrographs.

REFERENCES

1. Mohammed, C. F., and Schour, I.: Experimental Cavity Preparations in the Molar ofthe Rat, J. D. Res. 34: 429, 1955.

2. Maurice, C. G., and Schour, I.: Effects of Sodium Fluoride Upon the Pulp of the RatMolar, J. D. Res. 35: 69, 1956.

3. Perreault, G., Massler, M., and Schour, I.: Reaction of Odontoblasts to MedicamentsPlaced in Cavity Preparations in Rat Incisors, J. A. D. A. 52: 533, 1956.

4. Silberkweit, M., Massler, M., Schour, I., and Weinmann, J. P.: Effects of Filling Ma-terials on the Pulp of the Rat Incisor, J. D. Res. 34: 854, 1955.

5. Massler, M., Perreault, J. G., and Schour, I.: Experimental Pulpotomy in Incisors ofthe Rat, J. D. Res. 34: 429, 1955.

6. O'Malley, J.: Experimental Pulpotomies in Rat Incisors, Master of Science Thesis,University of Illinois, College of Dentistry, 1956.

7. Miyamoto, O.: Pulp Reactions Following Surgical Amputations in Rat Molars, Masterof Science Thesis, University of Illinois, College of Dentistry, 1957.

8. Fish, W. E.: Surgical Pathology of the Mouth, London, 1952, I. Pitman.9. Glass, R. L., and Zander, H. A.: Pulp Healing, J. D. Res. 28: 97, 1949.