Cell-mediated immune response to dog pulp tissue altered by Grossman’s formula sealer via the root canal Pulp tissue from three experimental dogs was tested for its antigenicity before and after incubation with Grossman’s formula sealer (GS).* Cellmediated skin-test reactions showed an Increased response to pulp that had been incubated in Grossman’s formula sealer. washed. and then injected as compared with pulp or GS alone. In vitro analysis of cell-mediated immune response (lymphocyte proliferation) showed a marked increase when pulp tissue was incubated in GS and washed as compared to saline-treated pulp. Comparing the saline-incubated pulp to the GS-altered pulp revealed that the increase in radioactivity was statistically significant at p ~ 0.0001 Therefore. Grossman’s formula sealer altered dog pulp tissue and rendered it antigenlcally active, and a specific cell-mediated lymphocyte response was produced.

1

n recent studies. Block and co-workers’ ’ have shown that dog pulp tissue can he antigenically altered by various endodontic pastes. sealers. or medicaments. With the USCof these antigens. immunologic skin tests. hemagglutination. and mixed lymphocyte reaction assays have confirmed that both the humoral and cellmediated divisions of the immune system arc responsive to immunization via the root canal. In addition, O~Ion animals immunized with biochemical studies”-“’ these altered pulpal antigens demonstrated that sifnificant increases in serum levels of lactic dehydrogcnase. alkaline phosphatase. and serum gamma globulin. specifically I@. occur. As a result of these previous findings. further immunologic evaluation of $cvtlraI cndodontic sealers appears warranted. Therefore. the purpose of this investigation was tcl determine whether dog pulp tissue became antigen-

callq altered when combined with Grossman’s t‘ormula sealer within the root canal and lvhether ;I specific cell-mediated lymphocyte pr-oliferation to this material re\ultetl. METHODS AND MATERIALS Test animals The experimental animals were tive healthy, conditioned male dogs, each weighing approximately 27 to 3 1 pounds. With three of the animals. the experimental material. Gro5\man’s formula sealer IGS).* was combined with the clog dental pulp: this complex acted as the antigen. 7‘11~ remaining two dogs scrvrd as controls. III the first control dog saline solution ~vas combined \h:ith hi\ clcntal pulp to tcht thi\ c~~mplex’s Lintigenicit); the \ccond dog ~3s LI& :I\ ii negative control for the cell-mediated skin tests. The animals were housed i~r Irooms with controlled humidity and alternating lighr and dark cycles The! were fed lahoratory dog cho\r XI libitunl. Sensitizing

method

fhc \tznsltiting

the procedure\

technique

&vcloped

used M as C~nloditication ol by Nishidu and associates ”

Number 4 Thefollowing procedures were performed on&bated dogsanesthetized with 10 to 12ml. of Suital.”All teeth were radiographed, scaled, and polished prior to rubber-dam isolation. Twenty caries-free teeth from

Antigen preparation for in vitro testing

Ineach experimental dogandthesaline control dog, the initial sampleof pooled pulp that had been frozen in 10 ml. of saline solution was divided into two 3 mg. portions. One portion in each dog served as a control; that is, it was not incubated with the Grossman’s formula sealer. In the three experimental dogs, the other 25 mg. portion was incubated with the GS material for 48 hours, washed, centrifuged, sterilized, sonicated, and concentrated with filters.* This antigen was designated as frozen pulp incubated with material and thoroughly washed (FPM). The necrotic pulp that had remained in all twenty teeth in each experimental dog after initial extirpation was removed from each of the five teeth before placement of GS at 7-. 4, 3 I -, and 28-day intervals; it was processed in the same manner. This tissue was referred to as necrotic pulp incubated with material and thoroughly washed (NPM). This necrotic tissue was incubated and treated in the same manner as the frozen pulp tissue (FPM). The amount of protein ofthe antigen was analyzed by the biuret test. and 1.50 nlg. per ml. of saline solution was used for in vitro testing. In the case of the saline control dog, the entire SOmg. portion was incubated with sterile normal saline solution for 48 hours. washed. centrifuged, sterilized, sonicated. and concentrated with a filter. The necrotic pulp that had remained in all twenty teeth in the saline control dog after initial extirpation also was removed from each of the five teeth before obturation at each of 7-. 14-, ?I-, and 28-day intervals.

each dog were used. A sterile endodontic technique was used throughout the experiments. The operative area was scrubbed with 5 percent Mercresin for 5 minutes before access to the pulp chamber was gained with a No. 4 round bur in a high-speed handpiece. The pulp tissue was extirpated with endodontic broaches. Hemorrhage of the pulp was controlled with cotton pellets. A thick layer of cotton was placed over the pulp. and the access opening was sealed with IRM cement.? Each dog was given intramuscular injections of SO0 mg. of penicillin and 2.50 mg. of streptomycin immediately’ after the operation and three times a day for the ensuing 3 days. Pulp tissue was removed from twenty teeth of each animal. Each animal’s tissue was separately pooled. homogenized, and divided into equal portions of 50 mg. Half of the pooled pulp of each dog was immediately frozen at 4” C. in 10 ml. of sterile normal saline solution. In three of the dogs, the other half was washed, centrifuged, concentrated, incubated with the experimental material, rewashed, and then mixed with 4 ml. of Freund’s incomplete adjuvant and emulsified. Four 2 ml. aliquots of this 8 ml. mixture were injected intramuscularly in the region of the right and left cervical lymph nodes and the right and left popliteal nodes. This primary immunization was performed on each of the three GS dogs and one saline control dog immediately after pulpal extirpation and material preparation. Each dog was injected with only his own pulp tissue, which had been previously extirpated and treated. At 7-day intervals for the next 28 days, the root canals of five of the teeth in each of the animals whose pulps had been extirpated previously were instrumented and filled with GS sealer. The access opening was sealed with IRM cement. At each of these four intervals of subsequent irnmunization via the root canal, the same procedure used in the management of animals and tissue antigen in the primary immunization was strictly followed. On each occasion, however, before the five teeth were filled with GS sealer the necrotic pulp that had remained after initial extirpation was removed and stored in 10 ml. of sterile normal saline solution and frozen for in vitro testing. With the control dog, the procedure used was the same as previously outlined. except that sterile normal saline solution was used instead of the rxperimental material. The fifth dog was used as a control for the cell-mediated skin test,

A standard technique for measuring cell-mediated immune response is the mixed lymphocyte reaction.“-” By labeling lymphocytes with tritiated thymidine. it is possible to measure DNA synthesis and thereby calculate the lymphocyte response to a specific natigen. Prior to extirpation (S,) and at the thirty-fifth day (Sa). whole blood was drawn from the external jugular vein of each of the three experimental dogs and one control dog and defibrinated by swirling with glass beads for 10 minutes. The blood was diluted I : I with normal saline solution, layered over a Ficol-Hypaque (sp. gr. = 1.074) gradient,“-” and centrifuged at 400 g for 40 minutes. The lymphocyte-pure band at the interface was aspirated and washed once with Hank’s BBS, and the cell concentrations were determined (5 X IO;’ cells per wall). Cultures were set up in Limbro plates. using medium TC-199 supplemented with 100 uU/e/ml. L-glutamine plus 20 percent fetal calf

*Surital, Park-Davrs Co.. Detroit, Mich. tIRM. L D. Caulk Co.. Milford, Del.

*Amicon Filter. Lexington. Mass

Measurement

of cell-mediated

immune

response

q Pulp mcuboted in solw only I Pulp ancuboted with G S ETI PHA posdwe control 4960

Plates

wcrc

atmosphere thymidine

of

additional glass

pure

fiber

paper

were

ncintillant

Gm.

POPOP

proton

for

+ 12 7-i Changes

G.S. DATA

REPRESENTS

AVERAGE

3

tissue

DOGS

(NPM)

pulp was

a~ntrol.

IZ:

Awraf~

raclioacti\

c‘ dim

ot

I)niphocqte5

in

rc’-

sponse to antifcns 01 (he fro/en and nc‘crotic ptiIr> of aalinc control dog and experimental GS dogs pulp bctorc incubation with GS material. Ncgliglblc radloactivit) indlcatcs lack of lyn’phoc! tc stimulation in response to unaltered pulp tia\uc antigens. q : A\ crayc dpm t11 stinlul;ltc’d lymphocytes 111 rcsponsc to antiyens 01 l’rolcn ;Intl necrotic pulp of three qxrimcntul Jogs ;lKtcr the> had heen altered by GS and thorotqhl! M ashed. There is ;I distinct increase in radioacti\ It)-lymphoq te pmliferation. M hlch indicates :I specific rcsponsc to altered pulp tissue antigens. Therefore. by cornparing radioactive uptake le\ cls 01. prolit’cratiy lynphocytes in ircsponsc (0 fro/en and nccrotlc pulps bcforc ;uicI after ulteration uith GS. there is il noticeable Inc‘rc;lst’ in Iymphocy~~~ prolif’ctxtion ~licn lulls i\ altc’rvtl I‘hc~ tl;u;i ~4i‘rc statist1 call! significant art p 0.000 I

solution

dermatly

shaved

each

Fife,

microliters

culture.

analysis:

The

from

of each

following

the three

antigen

antigens

experimental

was were

used in used

for

dogs the frozen

hacl\

ration,

manner of

in 0. I intra-

the experimental with

dog

to his own

in-

0. I of

into the cleaned that

had not

ken

pulp oi- an\’ cndodon-

observed

illuslrated taken

smelling.

indLk

and evaluated

in

af‘tel- the procedure.

da> I$)

in Fis.

from

whole

no activith. wcrc

to occur

schedule.

levels

The

of lymphocbtc

at the antigen

and necrotic

(FPM,

cul-

of the dogs

day of the experiment)

The highest found

the GS material

I represents blood

of immunization

of I : IO in both the frozen with

was

In addition.

of necrosis.

M’;IS

pulp tis-

;I material

intradermall

;I fourth

dog’s

and necrotic

as above.

data at S,, (the beginning

stimulation

back of’ each cx-

response

lymphocytes

control

of thcsc

and injected

The presence

The radioactl\it! on the 35th

each

in

saline

solution

each do” C 23. 4s. and 72 hours

tured

normal

of

pulp

aI 3” C.

was mixed

incubation

and cr).thema

RESULTS Cell-mediated

1112.

01

and NPM)

pulp tissue

imniuniLed

tic matcrinl.

in sterile

As ;I control.

S;II~C

and necrotic

and shaved

I c.c‘. of CS uas in.jected prcviotisl~

UCI’C

specimen

for 1X hours

saline

t’roLen

beforc

in the

showed serum.

dog.

sue alone

and

(FPM)

I .O

(FPM

normal

untreated

jected

tissue

into the cleaned

perimental

\\crc c‘ounttl

analyses

a 5 .I) mg.

three times

pulp tlssucb

ml. of sterile

own

dog.

and homogenized:

treated

vials

per 1111nute

qatistical

incubated

I ml. of GS uashed Fig. 1. The data rcprexent the Iradioacti\it! of DhA-labeled Iympht-rcjtes in r-esponse to the xtrioub antigens Lit ;I dilution of I : 10 All changes in disinttl~rat~ons per nunute (dpm) \ slut hnvc been c:dcul~~t~d b\ \uhtI-actin2 background radioactivir! of ;I blank saline control. n : The amounr 01‘ radioactive disintegrations per minute ot’ PHA. There is strong activitI indic;lting I\ mphoc) te stimulation and ;I pood positive

and

scintillation

skin test

his ou n fro/en OF

POP).

giass

TV 0.0.17!,

in disintegrations

For each er\perimental I IO

’ Fil-

counter

1).

Cell-mediated diluted

CF/C

harvesler””

I- toluc‘ne

in a liquid

and standard

l’or an

on M’hatman

(3.X

Gm.

IO minutes

(f;ig.

incubated

scintillation

was added

calculated.

performed

Antlgens

Jlcytide

tcl each culture.

added

an automatic

in liquid

monitor::~

were

carbon

Lverc

and harvested

using

placed

hials.

I .9) was

Iymphocytcx

10 hours

counted

95 percent

in

(sp, act. =

Cultures

ters

incubated

at 77” C. for 4 days and 1 uCi of tritiatcd

NPM).

pulps

dilution incubated

The values

of r;t

and necrotic pulp incubated with GS (FPM, NPM): frozen and necrotic pulp, incubated in saline solution. of these same experimental dogs before their pulps were treated with GS: frozen and necrotic pulp. incu-

dioactivit) uptake of DNA in lymphocytes are rcpresented LL\ change in disintegrations per minute (dpm) after the background saline control has been subtracted. These data \how that the frozen pulp before incubation

bated in saline solution. of one dog which was an independent control: phytohemag~lutinin (PHA) as a posi-

with GS had an average activity of 8 I2 dpm, the necrotic p~llp exhibited 689 dpm.

tive control

and

;I

culture

with

no antigen

(saline)

as a

negative control. All sample antigens were tested at the following dilutions for lymphocyte stimulation: I: I I:?. 1:5. l:lO. l:‘5. 1:X). I: 100. l:250. l:SOO. I : IO(K). and I : 1500.

Howevci-. radioacti\it)

when ro\c

the pulp was altered

whereas

by the GS. the

to 4.2 I6 dpm for fro/en

pulp (FPM)

Cell-mediated immune responseto dog pulp tissue altered by GFS 375

Volume 47

Number4 Table 1. Cell-mediated skin test data Necrosis

Swelling

Induration

Eryhemcr

3 GS dogs

Skin test: 48 hr. NPM FPM GS alone FP alone NP alone Skin teht: 72 hr. NPM FPM GS alone FP alone NP alone Saline- treated

dog

Independent

+ + -

+ + -

++ ++ -

++ ++ + -. -

f-t ++ + -

+++ +++ + -

+++ +++ + -

+++ +++ +I-

-

-

-

-

-

-

-

Skin test: 48 hr. NPM FPM Saline Skin tc\t: 72 hr. NPM FPM Saline Control

dog -Negative

-

control

control

-

-

(Not immunized to pulp or any material previously at 48 and 72 hr.) These data reprcvxt the average of the responses of the three experimental Grossman’s formula sealer dogs and the saline control dog. A = Mild reaction of 10.5 cm.; t + = moderate reaction >0.5 cm. but < 1.0 cm.; + + + = severe reaction of > 1.0 cm.; - = negative response.

and 3.9 19 dpm for the necrotic pulp (NPM). Comparing the saline-incubated to the GS-altered pulp indicated that the increase in radioactivity was statistically significant at p < 0.0001. The corresponding positive control of PHA gave dpm of 4,460 for the FPM and 4,2 14 for the NPM. To further verify these data, the frozen and necrotic pulp tissues of an independent control dog were incubated in saline solution and tested for lymphocyte activity. The average radioactive uptake for dpm of the frozen and necrotic pulps treated with saline solution were 774 and 941, respectively. The corresponding positive controls of PHA were 3,114 dpm for the frozen and 3,240 for the necrotic pulp. Comparing the saline-incubated pulp from the independent control to the saline-treated experimental pulp (FP. 812; NP 689), indicated that there was no statistically significant difference. Skin test-ceil-mediated

reaction

(Table I)

The cell-mediated skin-test data are summarized in Table I. The data represent an average of the responses of the experimental GS dogs and clearly demonstrate that in the experimental dogs at both 48 and 72 hours after skin injection, the pulp altered by GS (FPM, NPM) produced a more demonstrable swelling, necrosis, induration, and erythema than pulp in which the GS or pulp alone was injected. In general, the clinical responses were more severe at 72 hours than at 48 hours. In the control dog whose pulps were treated with saline solution and in the dog that had not been immunized to

his own pulp or to any endodontic material, there were negative skin test responses. DISCUSSION The cell-mediated skin-test reactions demonstrated that GS material alone produced mild necrosis, swelling, and induration, with moderate erythema, as compared to pulp tissue altered by the material and injected. In both the frozen and necrotic pulp altered by the GS there was an increasingly severe response of erythema, induration, swelling, and necrosis. The principal requirements of a positive cell-mediated reaction are the progressive development, over a 24 to 72 hour period, of erythema, swelling, and induration.‘“. I” This response may produce necrosis, can be as large a 6 to 8 cm., and usually subsides after 72 hours.“. ” The histologic response of this skin test is characterized by a tremendous accumulation of macrophages, which is thought to contribute to the induration of the clinical lesion.15, l6 Since neither the untreated frozen nor necrotic pulp alone was able to produce a positive cellmediated skin-test reaction. the GS material v~as implicated in altering host tissue antigens. Because of these skin test results, we thought it was important to determine whether a specific cell-mediated reaction was involved in this immune response. From our evidence (Fig. I), it is apparent that when both the untreated frozen and necrotic pulpal tissue from the experimental GS dogs and the independent control dog were incubated with saline solution the radioactive uptake-

Block et al.

376

lymphocyte

Oral SIX&. i2pril. 1979

proliferation

when

a portion

from

the experimental

GS and tested

dogs

proliferation

activity

was

statistically results

stimulation

to unlreated

ever.

when

with

lymphocyte effect

of

possible

tissue

tered

used in numerous Koch

nol alone

of GS. dental

that

various

since

evidence

from

skin-test

data. This

specific

tinely

used endodontic

filling

.“:{ and

topathology shown frestll) placed

Grossman’s tllixed

Muruzabal”’ reaction

of

Grossman’s

formula

to \Z’C

01‘ IXI~I~

to alter tls\uc

to vital

root

culture

and rcrl-

canal

studies’“-

”

hiss-

”

ti;i\~c

to be toxic.

U’hcn

~‘kttlLth

sealer

hcerl

scvert‘

In addition.

in studying that

has

intlanim;ltloii Erausquin

and

the periapical

had been

sealer.

waler.“’

periapical

healer tissue.

were produced. rat molars

tticii-

reaction.

itnplanta.22

formula

found.

\uppot?

is the ahilit!

is the

tissue

tis\uc

o\,erfillcd

that the degree

\\ ith

of intlan-

mation was dependent on the extent to which the rnatc rial had mixed with tissue dchri\ and fluid. Thi\ iiitlammatory

reaction

became

more

WWY

when

the

amount of tissue remnants increased and the ovcrlillcd mass uas less. These morphologic ob\crvatlons tcntl to support tcration

our recent of

necrotic

immunologic pulp

and lymphatic

transport

cndodontic

from

~~xtic

subscqucnt

tissue

finding\.

It is the aI

b> niarerials

\\hicti

stimulates an immunologic response ’ “’ Thus. the I\c) is the adequate biomechanical clcanslng of the root canal with the rctnoval of as much of the necrotic tissue

supply

there

animal

\aticln

of

MI

studies. .tpparcnt

spread and uith

tions.“”

w

have

materials

manner.

forms

an antigen.

contirm

that (irossman’s

accomplishctl.

Thus.

Irot

both quantitv

i)htain4 trcmcl!

placement

and

hecn \how II to alter

host tissue

call! active. this matcrial’x he qucstionctl

findings

can alter

formula

Before

pulp

quantlta-

t-csponsc.

an adequate

Sincr C;rossman’5 irritanl and cytotoxic,“”

in a suitable

sealer

is important.

tissue suggest

this study’s

of Grossman‘s

hou.evcr.

stages

and that pulp tis-

combined

formula

amount

used ill the Ioo( canal

at varying

“’ strongly

tissue and render it antigenic. 01’ the immune In m c\alu;~tion Iion of the exact

investigaHowever.

alone or. more likely.

when

c>t

out immunologic

are haptens

carrier.

process

findings.

Our data’

the

of material

In independent response

that endodontic

within

tication

not rule

dit-

obscrinteresting.

quantities

similar

does

ot‘ the material

and ’ “I

from

response

small

probable.

by the material.

se&~

action.

is quite

as seen histologically

sue. the protein

the

to recognirc

results

this lack of an Inflammatory

alteration

On

and Langeland’P

and detoxi

observed

i‘s-

(immunologic

particle\

I>,mph drainage

used. this is highly

however.

inflammatory

LL’ith the diffuse

nlq

treatment.

to correlate

IO sealer

no&s

sealer

It is important

Walton

can

and in-

of this clinical

11 is cliffcult

tercnt

canal

biochemical

are systemic

to

con

that endo-

loot

endodontic

ionsequences

tissue This

sho\ved

infrequently.

during

loreign

of the hema-pulpal

the

appointments,

I-LTU~I

In anitnal\

of vital

into

rhu:ill~ disappears.

inatcrial

iti-

to these

I” which

Not

in

no apparent

systemically.

significant

ape\

demon-

particles

the ability

introduced

the

they

this.

canal

pulp tissue.

sealer

III response

data’

matcriaI\

of

materials

performed a root

procedure,

illustrates

prciious

OLIY

recognition

&wmatl’s

in proximity

and necrosis

to

to Lt\aluatc

the Liettakcst tlnh in solid

intramuscular

stud!

have

radicular

Despite

14;IS present

togenous lirms

this

noclcx.

This

that

dCbridc-

heal and resolve

and placed vital

presence

particle\.

lymph

I”

standpoint.

techniques

using

11mph

Although

itntnunogcnii

\vhen one is Irytn?

material\

l’ollowing

biochemical)

eugc-

and :r~rw

assays

concept

antigenic.’

biologic

:I

i\

did not provide

it is difticult important

that the important

From

bc

alow

and immunologic

think

01

positive

ptutlucc

immunologic

a toxicologic

der that tissue

and

and associates”’

rcsponscs.

i\,

that

acknowledge

ma>

Koch

is especially

compare

full>,

st10wI1

regional

with

histologic

rcwlt iti x\x~cmicall! muiic~logi~~ ihangcs.

as \veIl as root c,;ulal ll;I\c’

minute the

teeth

contact

scparatcl!

cugetlot

of adequate

and Langeland””

monkey

tn

dontic

Ix This

hcc~at~sc

materials

We

materials

However,

Studies

and eugenol.

can act as an allcrfen

test reactions.

core

it i\

consideration

\Valton

in direct

One

because

complication.

Kcccntll.

Hov, cvcr.

;I cell-mcdiatcd immune to pulp tissue xl

and colleague\“’

skin

their

concentrations. V,C‘ ha\c

Ivithout

tlammation

I)\

proliferation

an important

sealers.

;I\

marked the

since

demonstrated

by the liquid

course,

altered.

cases that are overfilled

\tratcd

HOU-

se&t-.

not bc altered.

unlikeI>

response-lymphocyte

and

of the GS or tc\ting

or decrcasrd

highIS

and definitively

xc

l‘herefore.

merit. Nan\

sealor

occur.

ma)

that host tissue might

this appears

EAca/er

xs posslhlc.

pulpotomie\

O.OOOI

byproducts.

formula

the composition

increased

the I! III-

in radlt’-

at 11 .

antigens

proliferation

Bq altering

increase

canal

Grossman’s

with

;I lack of Iymphoc!tc

show& root

pulp

marked

in part agree with

which

pulpal

incubation

This

sirnilicanr

these tindings

associates”

incubated

potential.

resulted.

Houc\cr.

and necrotic

was

for its antigcnic

phocyte Therefore,

was not significant.

of this same frozen

this

technique

of material

scalei can

be

to cog must

hc

formula scaler is c\” because it has now and render

biologic

it antigen-

compatibility

must

CONCLUSIONS I

N’hen

i irossman’s

with dog pulp tissue, icall\, acti\
support.

nologic and

DI-. Allan data.

Dr.

Kaplan Daniel

t’or his aid in analyzing Green

financial

the immu-

for his aid in protocol

design,

Mr. Buddy Hutl~n and the rest of the staff of the Animal

Surgical tration

Rcwarch Hwpital.

Laboratwy. li)r their

McGuire

assistance

Veterans

in the care

Adminisof the dogs.

REFERENCES I. Bloch. R. hl.: AntlboJ> and Cell Mediated Immunity to Dog Pulp Tissue Altered h\ “N2” Paste Within the Root Canal, I. Dent. Rcs. 5& Special I\we A, Abel. No I. February. 1977. 2. Block. R. hl Lcwi\. R. D.. Sheat\, J. B.. Burke, S. H.. and Fawley. J.: Antihod) and Cell Mediated Immunity to Dog Pulp Tiqwe Altered h? Formocresol Within the Root Canal, J. Dent. Rcs. 56: Special Issue B, Abst. No. 758. June. 1977. 3. Bloch, R. M., Lewis. R. D., Sheats. J. B.. and Burke, S. H.: Antibotlq FormatIon to Dog Pulp Tissue Altered by N2 Paste Within the Root Canal, J. Endod. 3: 309. 1977. 3. Block. R. Iv.. Lewis, R. D., Sheat\. J. B.. and Fawley, J.: Ccl1 Mediated Immunity to Dog Pulp Tissue Altered by Formocresol %‘ithin the Root Canal. J. Endod. 3: 423. 1977. 5. Block. R M., Lewis. R. D.. Sheats, J. B.. and Burke, S. H.: Antibody Formation to Dog Pulp Tissue Altered by Eugcnol Within the Root Canal. J. Fndod. 4: 53. 1977. 6. Block, R. M., Lcwig. R. D.. Sheats. J. B., and Fawley, J.: Cell Mediated Immunity to Dog Pulp Tissue Altered hy 6.5 percent Paraformaldehyde Within the Root Canal, J. Endod. 4: 346, 197X. 7. Block. R. M.. Lcwla. R. D 1 and Sheats, J. B.: Immunologic Skin Te\t Responses to Dog Pulp Tiswe Altered hy Endodontic

377

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