The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats S. Kakehashi, D.D.S., 111.S., B.S.," II. R. Stanley, D.D.S., N.S., B.S.,** and R. J. Fitzyerald, Ph.U., X.S., B.S.,"** Rethesda, M d . N.LTIONAL IXSTITUTE OF DENTAL RESEARCH, NATIOKAL INSTITUTES OF HEALTH, UXITED ST.\TES PIJRLTC HE.\I,TII SERVICE

T he prohleiu of 1naintninin.g tllc vitality of a clinically exposed dental pull) by vilrious conservative procedures to encourage the clevelopment of dentinal bridging lias been the suhjcct of numerous investigations.'- The nnpredietahility of rt.sults obtained frol-ll tlncsc proceclures, however, has been the source of great consternation to most operative clinicians. In tllosc instances of successful man- agement of pulpal esposures, a considerable par t of the success has been at- tributecl to the cstraordinarjr degree of pulpal tissue resistance.R~easons given for the failure in fol-mation of a hard-tissue pulpal seal have included age of the patient, degree of surgical trauma, excessive sealing pressures, improper choice of medication, bacterial infection, and a low thrcslzold of host resistance.

Recansc of tlnc frequency of adverse results obtained after the pulp-capping treatment of acute or cllronically infected dental pulps, regardless of all the otlner conditions present, these particular cases arc no longer considered for this type of proceclnre. The microorganisms in these acute and chronically inflamed pulpal exposures 11ave therefore bcen thought to bc thc inlost siqnificant cause of failures in attempts a t dentinal bridging. To test conclusively the inflnei~ce of ~ i a h l c mic~.oorganisms on the fate of a surgically esposed dental pulp, a study utilizing germ-free animals was undertaken.

MATERIALS AND METHODS Thii-ty-six 7-week-old inbred Fisller rats, divided equally as to ses, were

nscd for this experiment. Tile group consisted of trvcnt,y-one germ-free animals

'Oral Medicine and Surgery Branell, Sat,ional Institute of Dental Research. "'"Chief, Oral Medicine and Surgery Rmncll, Sational Institute of Dent,al Researclt. """Chief, Gnotohiotics Section, Lalmr~tory of Jlirrohiology, National Institute of Dental

Research. 1.

Volurne 20 Number 3

Suryicrrl exposures of den ta l pu lps 341

and fifteen convention;~l control animals. Tlic germ-free rats were inaintairiecl in a. Reynicr germ-free system unit. Both the geiml-free and the conventional animals were fed, ad liI)itum, identical diets of pellet-form alltocl:~vcd Plusinn 5010 laboratory chow :~nd clistillcd watcl..

Tllc animals \vc3rc nncstllctizctl with il l1 ilitralwritonclal injcct,ion of pento- 1)arl)it;il sod i~~rn ( 4 mg. per 100 gram.. of 1)ocly weight). A earl~ide ~vound 1)n1. (size lh), n ~ o ~ ~ r i t ( ~ c l iri :L .~PTT.C~CI.'S spindlv-top1>ed hari(1 m;~~icli-c1, U S C C ~ to drill a hole through tlic occlnsal tliiclrncss of cnamcl and dentine of thc maxil- lary right first molar in order to cxpose the pulp tissne. A n attempt was madr to limit the exposure entry of the bur to the coronal portion of the tooth (Fig. 1, A and B)." In many instances, however, the crown was unintentionally penc- tratccl laterally or apically (Fig. I , C ) . No attempt was rnadc to restore or se

O.S., O.M. k O.P. L Septeml~cr, 196;

Fig. 1. A, S].ieciinen DK3-250-7-15, GFP44, So. 1 , 7 c l : ~ \ r s l)os(oli(~ri~.tive. Gern-free specimen s11on.ing int,entional exposure (arrow) of first. molar. ~ o i t l d(shris (F) 11% been forced into exposure and wen into pulp tissue. (Magnification, x40; reduced lh.)

B, Higher magnification of specimen shown in A . Food dchris (P) and dentiual spicules (8) can be seen within pulp cliamber. (hlagnification, ~ 1 0 0 ; reduced 1h.)

C, Specimen D63-253-7-6, GFC.44, No. 2, 8 days post,operxtive. Conventioilal specimen \vitll perforation penetrating through coronal 1)nlp c11arnl)er into i i~ torradi~ular area. De1)ris is on a level wit11 root apices. (Magnification, x3.5; rcbducecl 1h.)

the proliferation of epithelial rcsts of JTalasscz. Tllcsc prolifernting areas of epithelium coalesced to line the embedded sul).;tanccs, thus forming pciliodontal lesions and pockets. Peripheral sections of such lesions would reveal apical migrn- tion of the epitheli~un. Similar periodontal lesions also occurred in the germ- free animals (Fig. 7, ,4 and R ) . Germ-free experimental animals

Of the twenty-one gei~n-free animals, eighteen s u l ~ i r e d the opcrativc pro- cedures. Despite the pnlpal exposures and even the frequent gross pel-forations of the first molars, no completely devitalized pulp was observed i n any of the

li.

I Fig. d. A, Specinicn Dli8-2.52-5-15, GI!'(X4, No. 1, 8 days postoperative. Convcnt,ional

spccin~cn \\.it11 pulp cxpose(1 alld lingua.1 root visualizc'cl. S o t e ;junction of necrotic and vital tissues in 1ingu:~l root canal. ;\cute infla~nluatory cells cxn I)c ol)sc:rvcd infiltrating necrotic :1.11(1 vital tissues. (Magnification, x100 ; rcduced 'x.)

I) , Acljaecnt scction of specitnen sllolvn in A, ~ i ~ i l l ~ d l)y Gienisa technique to revcal colonies '*

of n~icroo~~ganisms in necrotic t i s s ~ ~ e . (3lagnificatio11, x450; rcducc,il lh.)

I

Fig. 8.

. - . , .." '

,. . .

I .a_ ~.

Pig. 3. S1)t:(:inl(~n T)(i: l-?ti l i-.)-L', (;Ia7(:44, S o . 5, 14 days yostol~t-rativc. i:otiventioni~l spr1t:i- nlcn demonstrating t+olrll~lcte pulpal 11c.crosis in hot11 roots wit11 apical al)scess fomnlations jar-

* row). (hragnification, x4O ; reduced % .)

Pig. 4. Specimen DB3-285-5-15, GFC44, No. 17, 41 days postoperative. Conventional speci- men stained hy Brown ant1 R r e ~ n l teclwiclur cxil i l~i t ing t,\vo colonics of microorganisms i n sof t tissues I,cyond a.piea1 f o r a n ~ c l ~ (ari.on-s j . (Magnification, x430 ; reduced 1h.)

CI germ-free animals. Tlle pulpal inflammation resulting from the exposure was minimal in every specimen. Not a single apical abscess was fonnd.

Dentinal bridging was already c ~ i d e n t a t 14 days, with prominent quantities I of matr is formation (Fig. 8, ailcl 3). The older specimens presented nlatris

formation complet~ly bridging or scaling the exposure. In every instance, the pulp tissue rcmaincii vital beneath thc 11e1vly forrnecl reparative dentinal bridge.

- The bridging occurred at any angle, rcgardless of the angle or severity of the exposure (Fig. 9, A and B ) . As postoperative time intervals increased, a gradual

O.S., 0.14. & O.P. L Scptcml~cr, 1965

I f I;.

F;g. ;T. .I. S I I ~ ~ ( ~ ~ ~ I I ~ ~ I I 1)(;:3-?S(i-(;-ll, GFC44) h-0. 18, 41 c1:1ys T J I I S ~ ~ ~ , ( > ~ : I ~ ~ V P . ( ' I I I I Y ( ? I I ~ ~ O I I : I ~ specin~en ~ r i t l ~ Iinir sll:lft protructi~lg Prom apical foranlen and surronrl~l(~c1 I I ~ leultocytcs. Arasses I of n~icroorganisn~s can l ~ c seen in root canal. (hlagriificatiol~, x100; rcilucctl lh.)

8, Sprrimrn 1)63-2S6-2-28, GFC44, XO. 20, 4 ' d ~ y s postopcratire. Coi~rentional specimen with necrotic pulp coi~nccting nit11 a1)sccss in pcriotlontnl ~ n ~ l n l ~ r n i l o 11y \ray of accessory canal. ( l l agni f ic~ t ion , x100; rcduccil lh.)

I J'

Fig. 6. Specilnrn D6:i-2Sti-14-5, (:T.'C-l4, No. IS, 41 tl:~ys ~)osfo~) i~ra t ive . Conventional spcr.i- lrlfw wit11 apical Iosion drrnonstrat i i~g typical foarr~ cells so ( . ~ ~ ~ q ~ ~ i ! i ~ t l y SC(?II ill llt1111al1 dental g r : l . ~ ~ u l o ~ ~ ~ a s . (Magnification, x440 ; rcduccd 1h.)

P constl*ictire ohliteration of tllc pulp c1laml)cr with rle\\l lrlatrix, containing Illany ccllular inclusions, n n s ohscr~ed (Fiff. 10, ./I ) . The oldest postoperat i~r qwci- Inelis demonstl.atc~cl matrix dcpositio~i f i l l i ~ ~ g tlic cuti~.c col'onal cl~anlbcr ancl root canals. S o ~ i ~ o t i ~ n c ~ s cl\-cln ;Ic.rc1s.;ory c.;111;1ls i l l t l ~ c i~p ic i~ l root arcs sllo\\c~l c.ol~st~.ic- m tiorl by 1nat1.i~ I'ol.iui~tio~l ill rcsl)oi~sc~ t o tllc occ1ns;ll exposure (Fix. 10, 11).

In cases in wliicll thc pulpal c1xposurc rcsultcd in scrcrancc oC a root, tllr pulp of the se~-crecl root retained its ~ i t n l i t ; ~ (F'ig. 11). \\'htn pcrfol-ations pcr- I rnitted the embedcling of food ancl clcbris deep into the periodontal tissues, tlic F resulting inflammatory response caused resorption of the roots e\rcn though the

Fig. 7 . .A, S ~ X ' C ~ I I I ~ I I (i:

O.S., 0.M. 6- O.P. I Scpt~ml)cr , 1963

Fig. 8. A, Sperinrt~r~ ( i : 3 O ( i : ~ O - 1 - 2 , C ~ l ~ ' I ' - l . l , Xu. :i. 1 1 ilayh l)ostol)c.~.:~tirc~. Germ-free specimen with food and debris in occ:lnsal cxposu1,e. Nacle:lr detail of surviving pulpal tissuc can he observed beneath bridge consisting of tlrntinal fra.gmcnts united I)p new matrix (arrow). I (Magnification, x100 ; rcduced %.)

B, Anot,llrr section of germ-free specimen shown in A , slzol\-ing matrix formation filling pulp chanlher. The stirnulus causes formation of rnatrix on all surfaces. A plug of fractured dent,ine can lw seen contril~uting to formation of the l~ridge. (Magnification, x100; rerlucecl %.I rn microorganisms in the pair of the exposed and severely damaged pulp. I n spite of severe surgical trauma complicated by impaction of food and debris, the germ- free animals exhibited a recovery and reparative response leading to dentinal - bridging. I n contrast, in the conrentional animals under similar conditions, thc situation deteriorated progressively from a n initial severe pulpal inflamlrlation to coinplete necrosis. Since i t is not possible to sap which of the many microbial L species involved were most responsible for the observed findings, it must be con- cludecl that present techniques for tlie clinical management of exposed pulps must be critically reviewed to include maximal measures for the eradication of

I all bacteria from the site.

SUMMARY The pm'posc of this study was to obscrve the pnthologic changcs resulting L

from untreated cxperimcntal pulp exposures in germ-frce rats as comparccl with

I Volume 20 Nurnlwr 3

Fin. 9. A, Specini(~ri G,?!)ti:;l-7-:3, lI:rsson, (41'1'44, Xo. 4, 14 clay:: postoperative. I n germ- free specilnen, (1c:spitc 1argc perforation in addition to pulp exposwe, pc~rpnnclicular I~ridging is apparent ( a r ro~v) . 3la. t l . i~ production is also occurl.ing on adjacent surfaces. (Magnificat,ion, x100; reduced 1h.)

B, Bpccin~cn 630636-10-4, GFP44, S o . 9, 32 days postoperat,ive. Tn germ-free specimen, note completion of brirlge with vital noninfltnncd pulp tissue remaining. Bridging can occur a t any angle. (Alxgnification, x100; reduccd %.)

conventional rats with a normally c o ~ n ~ ~ l e s microflora. The pulp tissues of thesc rats were exposed by drilling through the occlusal surface of the maxillary right fii-st nlolar with a car1)ide round bur mounted i n a je\~~elcr 's spindle-topped hand mandrel. After varying postoperative timc intervals (I to 42 days), the animals weye killed ancl the appropriate tissues were srrially sectioned.

B y the eighth day, 1-ital pulp tissue remained only in the apical half of the roots in the conventional animals. Cornplctc pulpal necrosis with granulomas and abscess formation occurred in all older specimens. Evidence of repair mas unifoi*mly lacking.

I n contrast, no devitalizccl pulps, apical granulomas, or abscesses were found i n the germ-free animals. Denlinal bridging began at 14 clays and by 21 and 28 days was comldete, of the angle or severity of the exposure.

These rest~lts, even in the face of prosy food impactions, indicate that the

O.S., 0.31. tt O.P. I September, 1963

I.'i(/. 1 0 . .At Slwcinrc~li 6:1063S-12-3 Jlasson, GFP44, So . 11, 32cI :~ys postopc~rntivc!. .In gcrm- free specimen, note obliteration of coronal pulp cl~aml)cr I)y nlatris i'ol,mation : ~ n d nu1nl)cr of laeunao from ccllalar inclusions n-itl~in matrix. (31agnification, xl0O; rotluced %.)

I{ , Spccirncn 630635-19-6, GFP44, No. 8, 32 days postopcrativc. I n gerrrl-freo specimen, gradual ohl i te r~t ion of pulp chamber and root canals has occurred. IIere ol~literxtion of root I canal is evident, Icacling to con~triction of a n accessory canal. Xote many ccllulxr inclusions. (Magnification, x100 ; rcducetl x.)

Fig. 11. Specimen 630632-8-4, GFP44, So . 5, 14 days postoperative. Germ-free specimen exhibiting severance of root. Se~er theless , remaining pulp tissue in scrrrctl root remains vital.

II Note dentinal f ragmcnt ,~ being coated ~ r i t l i new nintrix. (Magnification, x100; rcclurccl %.) Fig. 12. Specimen D63-2.71-11-11, GFP4-1, No. 2. 7 claps l~ostopcrativr. Gorm-froo specimen

in wl~icli perforation has occurred, permitting foocl and dcl)ris to be channeled into deeper periodontal tissues. Resulting inflammation lias led to root resorption, even though pulp in root canal remains vit,al. As root is resorlwd? new alveolar I,one is being deposited (arrow) to I co~npensate for ciecrease in root size. (3lttgnificatior1, x100; rerluccd lh.)

presence or absence of a microbial flora is tlic major determinant in tllc healing of exposed rodent pulps. c

REFERENCES 1. Rapopcrt, L., and Aljramsou, I. I.: .ipplication of Steroid Hormone.: in Pulp capping nnd

rulpotomy Procedures, OKAL 81-nc., ORAT, h l ~ n . 6 ORAI. PATH. 11: 345-548, 1938. I 2. Rosmstcrn, S. S.: Pulp Capping 111 Deciduous 'l'cctll: Rcport of a Ten-year Study, J. Am.

Dent. A. 29: 1632-1643, 1942. e

3. Z ~ l ~ d e r , H. 8.: React,ioll of the P u l p to Chlciurn EIydroxidn. J. D. Kcs. 18: 3i:l-379, 1939. 4. Zarlrirr, H. d., and Glass, h'. L.: The I-Iealing of Phenolizcd Pu!p Exposures, ORAL SI;RC.,

ORAL NED. rY. ORAI, I'AT~I. 2: 803-810, 1949. 5 . Ellis, li. G.: Tllc! Classificat,iol~ nnd Treatrncnt of 11~juric.s to 7'c(>tI1 O F CI1i1d1.cn, etl. 4,

Chie:~go, 11160, Year liook I 'ul~lishers, 711c. ti. Rol~~uie r , I( . F., O s t r a ~ ~ d r r , 1,'. I)., nrld Cvo~rlcy, 31. C.: Clinir:ll Eudodont,ics, Pl~i ladclphin,

1RS(i, TV. R. Saul~tlers C o l l l ~ ~ a ~ ~ g . 7. I3olir1g, r,. I