0022-1554/90/$3.30

The Journal of Histochemistry and Cytochemistry Copyright © 1990 by The Histochemical Society,

Vol.

Original

Immunohistochemical and Proteoglycans Rat Incisors’ MINORU

TAKAGI,2

ATSUHIKO Department Oral

ofAnatomy,

Biology,

Received

June

FIROZ

University

ofAlabama

for publication

HISHIKAWA,

and Nihon

University

PG (C4-S,

School

School

16,

1989

ofDentistry,

ofDentistry,

and

Tokyo

University

KS, DSPG,

C0-S,

in revised

and

ofthe

type

and

(GAG)

for understanding

drates

in the

electric

form

C6-S)

repeating

charge.

enables them with calcium

tin

These

large

matrix

is a prerequi-

of GAG

molecules

hydrophilic

they

matrix

PG have and

been

dentin

The

carbohy-

carry

negative

associated

and

with

polyanionic,

which

cationic material; they interact phosphate in a stable colloidal influence

not

only

the

but also mineralization

organi-

of the den-

qualitatively

isolated

and

biochemically

(4,10,15,24,25,28,34,43,46)

and

quantitatively

diography

utilizing

SO4

thesis

secretion

ofsulfated

and

chains

to attract water and and maintain calcium Therefore,

dentin

and

(33,34).

and

in predentin

differ

and

(PG)

in dentinogenesis.

polyanionic

are highly

of the predentin

GAG

role

disaccharide

(2,3,5,44,55).

zation

101, Japan

September

in preden-

ofproteoglycans

in predentin

their

the PG core protein

state

distribution

GAG

identified (Table

between

has also been

these

employed

sites.

to study

by odontoblasts

HOSOKAWA,

into

1) and

Autorathe syn-

(M7HH,YH,AK),

ofAlabama 6,

accepted

tin and,

in part

by NIH/NIDR

RR 05330 (FR). Correspondence to: Minoru omy, Nihon University School Chiyoda-ku, Tokyo 101, Japan.

2

grants

‘ilkagi, DDS, of Dentistry,

DE 08466

and

September

to a lesser

the

Alabama

extent

Department

35294

15,

1989

(C4-S

and

of

(FR).

(9Al718).

small

DSPG),

in the

dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a defitrite difference between PG ofpredentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules

during

(J

Histochem

KEY

WORDS:

Dentin;

Mineralization;

and

dentinogenesis

Cytochem

and

38:319-324,

Predentin;

mineralization.

1990)

Proteoglycan;

Glycosaminoglycan;

Immunohistochemistry.

dentin

using

cationic

dentin

GAG

scopic

levels,

combined

(18,30,47,53). reagents at

A variety have

the

light

been

ofcytochemical

applied

to

tified

using

various

ofGAG

in predentin

recently

developed

GAG

The

and

present

study

the

utilizing

five MAb

recognize

unsulfated

as well

of predentin

and

may

allow

studies have not the precise nature

dentin

can

inbeen and

be directly

antibodies

and

and

dentin

iden-

(MAb)

to

was undertaken

distribution

one polyclonal

chondroitin

6-sulfate

as large

methods

digestion

monoclonal

immunohistochemical

chondroitin

micro-

PG.

identify

5),

and

and

electron

cytochemical

enzyme

direct demonstration of GAG types, such performed to our knowledge. Alternatively, distribution

predentin

and

Although

GAG-degrading

techniques

stain

(16,17,31,36,49,54)

(1,19-22,37,38,48).

with

small

antibody

(C0-S),

(C6-S),

of

and

dermatan

GAG

which

chondroitin

keratan

sulfate

sulfate

PG

to

and

PG

specifically 4-sulfate (KS)

(C4-

(7,8,12),

(DSPG)

(42).

preden-

and

Materials 1 Supported

and

at Birm:ngham,

1989;

tin

glycosaminoglycans

site

Article

YASUNOBU

Introduction Knowledge

pp. 319-324, 1990 Printed in USA.

3,

RAHEMTULLA

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aidehyde fixation of rat incisors using (a) four monodonal antibodies (1-B-5, 9-A-2, 3-B-3, and 5-D-4) which recognize epitopes in unsulfated thondroitin (C0-S), thondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatinent of tissue sections with chondroitinase ABC localized the majority of stainable

No.

Localization of Glycosaminoglycans in Predentin and Dentin of

HIDEKI

KAGAMI,

38.

Inc.

BRSG

Tissue Preparation.

2507

PhD, Department of Anat1-8-13, Kanda-Surugadai,

Methods Twenty

Wistar

rats,

weighing

each, were used in this study. The upper jaws were fixation with modified Karnovsky’s (29) fixative maldehyde-0.5% mm,

and

were

glutaraldehyde further

immersed

approximately removed containing

in 0.1 M cacodylate in the

same

fixative

buffer,

after

100 g

perfusion4% parafor-

pH

7.3, for 10

for 50 mm

at 4’C,

319

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

320

TAKAGI,

Table

1 . Biochemical

studies

of GAG

Animal

Predentin

species

Rachitic

in predentin

and

Proposed

puppy

Dentin

a Abbreviations

acid,

HA. b Identified

and then They

GAG

several

Human Human

C4-S, C6-S, Chondroitin

with

constant

HA

(C6-S

HA, DS, CO-S (C4-S sulfates, HS

cal one

third

of the

Rat

C4-S

Smalley

and

Rat

C4-S

Rahemtulla

chondroitin, order

CO-S;

of their

(5-mM and

and

4-sulfate,

buffer

in 0.5

(pH

concentrations

thoroughly

7.3)

M EDTA,

chondroitin

pH

at 4’C. 7.3,

dentin

fluoride)

rinsed

maxillary

located

at the

incisor

were

6-sulfate,

in Table

con-

tamed

of benzamidine

phenylmethylsulfonyl

then

C4-S;

nation

ofantibodies,

Table dental

2.

of GAG. as well

labial

in 0.1

surface

examined

The characteristics,

as protocols

used

for

within this

the

2. MAb (Miles

specificity,

in the present

bovine

Chondroitinase

ABC

1-B-S

with

sclera

either

staining

study.

Second

nize

DS;

rabbit

3-B-3,

Heparan

and

Naperville,

technique

using

antigen

and

Hjerpe

(15)

(24) Embery

(46)

et al.

sulfate.

are listed

epitopes

nal antibody

with

protease-free

digestion (7,8)

and

HS;

(43) hyaluronic

PG (7,12).

MAb

on the protein

a nd light

were

remain

5-D-5

and

the

im munostaining

MAb

with Immuno-

was evaluated

as these

to the 5-D-4

the

of specimens

AC II (7,12).

polyclonal

DSPG

by injecting

digestion

ABC,

attached The

core of the large

micr oscope

in rabbits

study

II)

by Coster

Immunostaining

the prior

from

(DSPG

described

raised

II (42).

chondroitinase that

DSPG

the procedure

ob-

was prepared I) isolated

small

in the present

stubs

commercially 5-D-5

(DSPG

the

or chondroitinase used

ofthe the

were MAb

DSPG

In addition,

ABC

antibodies

5-D-4

IL), while the large

(42).

these

oligosaccharide

KS PG

antibody

9-A-2,

chondroitinase with

and desig-

study,

1-B-5,

as the

ter treatment

api-

Affinity-purified

1

sulfate.

were extracted from bovine sclera using and Fransson (11). Polyclonal antibodies a highly purified preparation of DSPG MAb 1-B-5, 9-A-2, and 3-B-3 relies on

Specifically identified GAG and

Antibody designation and class

dermatan

Laboratories;

by the hybridoma (40),

M cacodylate

Conditions for use ofthe MAb and the polyclo pulp, odontoblastic layer, predentin, and dentin”

Enzyme used prior to immunostaining

C6-S;

(43)

amount.

in 0.1 M cacodylate at 4’C

chondroitin

(15)

Jones and Leaver (28) Branford White (4) et al.

enzymatic

Immunostaining

(10)

et al.

Hjerpe

inhibitors

and

et al.

Clark

Engfeldt

buffer (pH 7.3). The demineralized specimens were cut into small blocks, each containing teeth with their surrounding periodontal tissues. Subsequently, the specimens in 0.1 M cacodylate buffer (pH 7.3) at 4’C were cut transversely into 15-sm sections by a Microslicer (Dosaka EM; Kyoto, J apan). These tissue sections were kept in the same buffer at 4C. Odonpredentin,

predominates)

Hjerpe

Rahemtulla

C4-S>C6-S>DS1’

iodoacetamide,

toblasts,

predominates)

C4-S>C6-S>DS’

for 40 days

agitation,

C4-S,

and (35)

Puppy

rinsed

protease

hydrochloride,

Linde

C4S>C6Sb

unsulfated

demineralized

References

Engfeldt

C6-S,

in decreasing

thoroughly

were

taming

used:

RAHEMTULLA

identified

C6-S>C4-S>DS’

Human

puppy

GAG

C4-S>C6-S>HA>DS>KS”

Rat

Rachitic

KAGAMI,

dentina

analyzed

Pig

HOSOKAWA,

HISHIKAWA,

recog-

protein

after

crossreact

with

core will

af-

MAb

antibody

(DSPG

I) and

of GAG

and

recognize the

small

PG in

PG

types

References

CO-S

(7,12)

Odontoblast

Pulp

Predentin

Dentin

3

3+

0

3+

4+

4.b

l

Y

0

34+

5+

4+b

anti-mouse

IgG1-HRP Chondroitinase

ABC

9-A-2

Affinity-purified

IgGi

rabbit

DS

(7,12)

5

Large

DSPG’

(42)

5

Small

DSPG’

(42)

3-4k

CO-S

(7,12)

Y

(7,8)

3

C4-S,

anti-mouse

IgG1-HRP Chondoitinase

ABC

5-D-5’

Affinity-purified

1gM

rabbit

Polyclonal IgG

Affinity-purified goat anti-rabbit IgG-HRP

3-B-3

Affinity-purified

1gM

rabbit

anti-mouse

IgM-HRP

Chondroitinase

ABC

antibody Chondroitinase

ABC

C6-S,

0-1

0

anti-mouse

IgM-HRP

Chondroitinase

a Staining b Intense

ABC

5-D-4’

Affinity-purified

IgG

rabbit anti-mouse IgGi-HRP

intensity graded 0. no staining was limited to C Enzyme pre-treatment of tissue d MAb 5-D-5 and the polyclonal

staining; dentinal sections antibody

KS

U, very weak staining to 5, intense staining. tubules, with weak staining in the remainder is not essential to 5-D-5 and 5-D-4 antibody against DSPG recognizes only the protein

of the

dentin

3+

3+

matrix.

staining. part

of the

molecules

and

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

not

the

carbohydrate

portion.

0

PROTEOGLYCANS

DSPG

IN

(DSPG

(42).

II), respectively,

When

using

digestion

with

study

Grand

Tris-HC1

were

NY),

The

sections before

indicated. azide (32).

for

and

peroxidase

(HRP)-conjugated

anti-mouse

San

Francisco,

ough

CA)

rinsing

with

previously

DAB

(23).

HCI buffer

The

(pH

Sigma

Chemical

added

immediately buffer

mounted

for light

mal mouse

2). The

antibodies

the

and

10 mm

consisted

7.6), dissolved

with

50 mg

Co.,

MO)

to which

at 4’C,

dehydrated

with

predentin,

in

for 20 mm

slight

decrease

tioned

sites.

all

staining

third

study,

of the

the dental

located

H2O2

were rinsed

with 0.05

cleared

was M

in xylene,

and

preparations,

for antibodies

tooth,

were

sections

5, C4-S, droitinase

DSPG, C6-S, and ABC pre-treatment

the

revealed

1-B-S antibody,

dental

pulp

labial

examined.

of 15-tm

The

pulp,

at the

nor-

specific

odontoblastic

layers,

surface

the

Light

within microscopic

immunoperoxidase

for PG.

strongly

tin

and

layer

parts

ofdentin,

(Figure

virtually processes, to

intensely 2a).

and

ABC

odontoblastic

weakly

The

S.D-S

stained predentin

the pulp

and

or

not

at

all

after

intensely,

moderately,

stained

chondroitinase

no staining

chondroitinase staining of the

no staining

with

matrix

5-D-4

chondroitinase

antibodies

the

showed

in the pulp,

was

not

observed

odontoblastic

or

mouse

negligible,

chon-

serum

very

layers,

was a

aforemen-

antibodies

normal

6).

there

of the

sites to

layer,

(Figure

ABC

aforementioned

in which

ABC

staining

staining

exposed

was ob-

the odontoblastic

lacked

antibody

not

Immunohistochemical

C4-S,

C6-S,

cific

for

neous

the

preden-

odontoblastic

of the

dentin

was

odontoblastic

dentin

matrix

layer

small

DSPG,

layer was identified

pre-treatment very weakly,

and

in the den-

has

ofPG

was

faint

and

and

study

back-

predentin.

tubules

or no fied

C4-S

studies and

C6-S

extract,

which

majority

of demonstrable

strating

studies

However,

only

definitive

and

in the den-

with

dentin

matrix. dentin

minimum Previous

have

demonstration

of

is consistent

with

the

of GAG GAG

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

the

bio-

demon-

(15,25,34,41,43)

amount of

identi-

vs the dentin

in the could

accomplished with the methodology employed in previous whereas immunocytochemistry allows direct visualization differences.

and small

of GAG

processes,

The

identify

exthe

component

and

species

in the

(18,

to a lesser Whereas

in the predentin

C4-S.

localization

large

and, matrix.

in predentin

other

decrease

not

distribution

of the

GAG

of rat and

a significant

(C4-S,

of rat predentin

contained

studies

odontoblasts,

could

reactive

of iOi

The

GAG and PG, with

homogeneous

in a ratio

teeth.

studies

in the predentin in the dentin

remainder

(43)

ofrat

specific

odontoblastic

in the

spe-

in the odontoblasinto

macromolecules

surrounding

biochemical

these

localize

reveals an intensely

reactivity

dentin

CO-S.

a heteroge-

autoradiographic

however

has a relatively

PG, the dentin

for

antibody

localized

incorporation

Our results

ofdemonstrable

predentin

and

previous

DSPG, KS, CO-S, and C6-S) tent (C4-S and small DSPG),

specific

of PG staining

SO4

dentin;

MAb

a polyclonal and

in predentin

with

GAG types.

five

and

characterized

in the present

predentin,

with

DSPG,

demonstrating

specific

tin.

ABC

large

is consistent

chemical

appeared

staining

KS, and

distribution

tinal

2b).

the odontoblastic but

predentin

stained

surrounding

of the (Figure

and

stained

in dentin (Figure 1). chondroitinase ABC

staining

tubules

remainder

rest of the

Discussion

the majority

of CO.

treatment,

strongly

moderately

the

antibody

examination

layers

the pulp,

the strong

to dentinal

whereas

stain

stained

However,

restricted

one

KS in rat teeth with or without chonofsections (Table 2; and Figures 1-6).

moderately, whereas no staining was visible The 9-A-2 antibody in combination with pre-treatment

predenapical

localization

after chondroitinase

with weak

to chondroitinase

specimens

staining

with weak to be their

in the

whereas

the pulp,

dentin

exposed

of the

for specific

30,47,53) dentin,

stained

specimens

substituted

ground

the predentin,

the

Control

tic

and

was identified

after pre-treatment the pulp, with very

not

droitinase.

in

ofthe staining were very and dentinal tubules sur-

4).

in the

control

was patchy;

M Tris-

Results In the present

no staining

but

In the specimens

observation

tin,

but (Figure

ABC parts of

(tetrahydrochloride;

For control

or BSA was substituted

matrix

Similar

as described

0.1 ml of 5.2%

ethanol,

examination.

H2O2

branches,

in

incubated

at 22’C,

of DAB

then

pre-

to strongly,

layer

processes showed strong staining, and filamentous material presumed

and

thor-

of 100 ml ofO.05

use. All tissue sections

microscopic

serum

without

for

medium

were

lateral dentin

IgGi,

2). After

odontoblastic ofgranular

and

Laboratories;

(Table

sections

solution

solution

St Louis,

BSA

layer moderately

moderately

so

enzyme

but only weakly Staining was not

chondroitinase the pulp and

odontoblastic

pre-treatment

1

anti-mouse

the

goat

to horseradish

(Zymed

over

dentin (Figure 5). antibody in combination

ac-

were

reaction

layer and

in S-PBS

specimens

and the odontoblastic

the

served in the The 5-D-4

S-PBS

without

the pulp, predentin.

or without intensely,

odontoblastic

0.1%

diluted

at 22’C

(rabbit

tissue

substrate

with

anti-PG

IgG)

in S-PBS

PBS,

substrate

mm

for 60

anti-rabbit

substrate

before

Tris-HC1

(Table

and (DAB)

the complete

a briefrinse

at 22’C

i:so

S-PBS

After

antibody

antibody, stained

diges-

or normal

5-D-5

The 3-B-3 ABC, intensely

BSA)

and

rinsed

serum

or goat

goat

diluted

3,3’-diaminobenzidine and

and

rabbit

mouse

second

1gM,

BSA was

peroxidase

thoroughly

with exposed

as well

H202

The

dentin.

strongly,

reactivity

and

the

although

rounding

chondroitinase 0.3%

dentin

specisaponin

(51).

endogenous

were

BSA.

0.03%

in

(BSA)J

antibodies,

of 1% (S-PBS

containing

to normal

and

and

the

3).

the dentin, the intensity and distribution similar to those with the 9-A-2 antibody,

[0.2 U/mI

elsewhere

to eliminate

M

for other

with

with

with and without

sections

treated

BSA

permeabilized

of

aldehyde

albumin

was omitted

solution

at 22’C

for 60 mm

and

treatment

to a protease-free

serum

at a concentration

these

in S-PBS

BSA

in S-PBS

bovine

as recommended

at 22’C

were

Labora-

free

(Figure

similarly intensely stained the odontoblastic layers and

The polyclonal antibody with pre-treatment stained predentin

acetate-O.1

1 hr at 37’C

incubation

the S-PBS

30 mm

i:io

diluted

in S-PBS

then

rinses,

for 30 mm

for

digestion

solution

with

the specimens

rabbit

visible

after

in 0.1 M sodium

1 mg/ml

during

Subsequently,

exposed

rinsed

protease-free

Kogyo Co; Tokyo,Japan)solution

The tissue sections

treated

and

to quench

matrix

in the present

PBS (Gibco

before

for 30 mm

exposed

were

and

to the S-PBS

were

BSA,

were

the consecutive

also added

serum

prior

treatment stained

with

in Dulbecco’s

washed

chondroitinase

tissue

as during

and

however,

tin

tissues

antibody,

treatment

at 4’C

pH 7.3, containing

in PBS (S-PBS)

tivity

7.3,

briefly

and

As a control,

sodium

321

in soft connective

the polyclonal

after

in PBS

ABC (Seikagaku

in 0.1 Tris-acetate,

non

pH

were

(7.3)

chondroitinase

when

DENTIN

is not required;

rinsed

glycine

Sections

buffer

mens.

and

evaluated

thoroughly

in 0.1%

(50).

(12).

are present

5-D-5

glycosidases were

Island,

sections

groups

and

AND

ABC. sections

tories;

which

5-D-4

specimens

chondroitinase

these

MAb

the specific

all the

Tissue

PREDENTIN

RAT

not

denbe

studies, of these

322

TA.KAGI,

Figure 1-6. Demineralized rat incisor after pre-treatment with chondroitinase

HOSOKAWA,

HISHIKAWA,

section stained with antibodies to CO-S, C4-S, DSPG, C6-S, and KS, followed ABC. D, dentin; Pd, predentin; Od, odontoblastic layer; P, pulp.

Figure 1. 1-B-5 antibody specifically stained tin stain moderately; and the dentin matrix

CO-S in chondroitinase is unstained. Original

ABC-treated magnification

section. The pulp stains moderatelyto x 135. Bar = 100 tm.

KAGAMI,

by peroxidase-conjugated

strongly;

odontoblastic

RAHEMTULLA

second

antibodies

layers

and preden-

Figure 2. 9-A-2 antibody staining showing the distribution ofC4-S andr OS in chondroitinase ABC-treated section. The antibody stainsthe pulp intensely, predentin and dentin strongly, and the odontoblastic layer moderately. In the dentin, strong staining is closely associated with the dentinal tubules (arrows; enlarged in b) surrounding odontoblastic processes, and both granular and filamentous material (arrowheads), presumed to be the lateral branches of the dentinal tubules, exhibit weak reactivity. Original magnifications: a x 135; b x 430. Bars: a = 100 sm; b = 10 rim. Figure toblastic

a

After pro-treatment layer very weakly,

with chondroitinase ABC, 5-0-5 antibody stains the protein core ofthe and predentin moderately, whereas the dentin lacks staining. Original

large DSPG. magnification

The antibody stains the pulp intensely, x 135. Bar = 100 sm.

the odon-

Figure 4. The polyclonal antibody staining showing the distribution of the small DSPG in chondroitinase ABC-treated section. Staining is visible in the predentin and dentin. In the latter site, strong staining is confined to the dentinal tubules and weak staining is also present in both granular and filamentous material, as shown in Figure 2b, whereas the remainder of the dentin matrix lacks staining. The pulp and odontoblastic layer are moderately to strongly reactive. Original magnification x 135. Bar = 100 tm. Figure in the

5. After chondroitinase ABC pro-treatment, odontoblastic layer, and weakly in predentin,

Figure 6. After chondroitinase ABC pro-treatment Original magnification x 135. Bar = 100 tm.

3-B-3 antibody specifically stains C6-S and CO-S. The staining is observed whereas the dentin lacks reactivity. Original magnification x 135. Bar 5-D-4 antibody

moderately

stains

KS in the pulp, odontoblastic

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

=

intensely in the pulp, very weakly 100 tm.

layer, and predentin,

but the dentin

lacks staining.

PROTEOGLYCANS

Previous

IN RAT

ultrastructural

have suggested

agents

from

removal

ing

of at

ofGAG

least

with

loss

AND

cytochemical

studies

the loss ofGAG

during

some

The present

(48). sistent

that

PREDENTIN

dentin

GAG

in dentin

results

species fixation

although

mask-

no GAG

using

staining

mineralization,

could

not

be

excluded

study

using

immunocytochemical

rather

than

masking,

323

DENTIN

as the

cationic

re-

as a possibility

DSPG

methods

alized

stain

is con-

is not

directed

tion

(see Table 1), are extracted and EDTA demineralization, and

PG staining

staining.

we have

for C4-S

that

EDTA,

ofwater-soluble

such

reten-

(45),

distribution ofthe mens (unpublished

stained material is very similar in the two speciobservation). Therefore, we assume that if PG

phosphate

extraction

occurred

during

not

the

(13,27,44,52),

dation

ofPG

esis

is consistent

ing

the

distal

tein.

However, comes

of PG

of these

tissues

(40).

The

flicting

The

study

to

molecules,

as the with

is presumably

ofthe

process

dentinal

staining

of this

ization

indicate site,

staining

then

agents

polyclonal

The

observation the

of the

dentinal

structural studies Previous light demonstrated

in Safranin alization not

is limited

exclude

as previously

the

(48).

tubule

C4-S

by prior possibility

identified

the demonstration in the present

ate to intense

demineralization

it would

reported

in the

present

localizing

C4-S, C6-S, observation

cal studies

(14) demonstrating

dominant

GAG

ofPG in the pulp was not the prithis tissue demonstrated moder-

study,

immunostaining

ety ofPG (CO-S, other sites. This

that

fraction

larger

amounts

of a van-

KS, and DSPG)when compared with is consistent with previous biochemichondroitin

of rat incisor

sulfate

was the pre-

pulp.

Acknowledgments terat

Dr Richard

T Parmely

San Antonio,

manuscr:pt.

Texas) for

We also thank

however,

in cartilage aldehyde some

by biochemical

wall

space

Lack

(University

helpful

ofTexas

comments

Dr Masaaki

Health

Science

andcritically

Toda for

Cen-

reading

his technical

the

assistance.

The

for the

known

DSPG

for the material

and

tissues fixation. PG

require

studies

fixatives

in

EDTA

ultra-

(18)

have

solutions

rat predentin and that a decrease by EDTA Therefore,

present

studies

at pres-

dentinal tubin the lateral

this will

2. BownessJM: cification.

5. Campo

deminer-

H: The formation

White from

ofground

WF: The surface chemistry of cartilage. 3 Biol Chem CJ: Molecular human dentine.

organization Arch Oral

RD: Protein-polysacchanides

disease.

and mineralization

Clin

of den-

1966

Present concepts ofthe role Clin Orthop 59:233, 1968

4. Branford teoglycan

Orthop

substance

of bone. 193:243,

V. The 1951

of heparan sulphate Biol 23:1141, 1978

of cartilage

68:182,

in cal-

and

bone

iron

pro-

in health

1970

RD, Betz RI: Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA).

6. Campo

previ-

staining

Cited G, Nakahara Rec 156:303,

1. Bevelander tin. Anat

and

of mineral-

structures.

is not

small

Literature

mineraliza-

accounts

matrix, except of the reactive

that

the

cytochemical studies that of this site for cationic re-

and

of aldehyde

ofPG

sur-

and

of tubular

35S04-labeled material from and Betz (6) have demonstrated

0 staining

Although objective

3. Boyd ES, Neuman binding properties

stain-

immediately

for confirmation. microscopic autoradiographic

the ability

to extract some dentin. Campo

predentin

tubules;

present

The

inhibits

its significance

of weak

the remainder of the dentin ules, may indicate the presence branches

PG

retention

However,

observation

small

ofperiodontoblastic

and ultrastructural an increased affinity

(17,48,49,54). The

the

in the

space)

stainable

dentinal

ob-

are common

molecules. space

and

tubules

and

used

ofthe

narrow

that

of the wall of the

EDTA

differences

the

con-

previously

PG

periodontoblastic

for

study

C4-S

which

antibody core

the

in

of these

been

of dentin

facilitate

ous light microscopic have demonstrated

not

epitopes

as proposed

would

has

have

in

previous

mary

We thank

to differences

the

aqueous

staining

whereas

in a net

difference

the dentinal

cores

(the

tubule.

may

with

the

located

the

for

of intense

association

protein

heavy

protein

The

and

tissues the

link

evidence

relative

EDTA,

elucidated.

processes

that

showed

significance

be

obser-

of epiphyseal

and

related

pro-

present

have

is no

demonstration in close

likely

crossreacted

rounding

ent.

physiological

of soft connective

to the

the

protein

study

and

odontoblastic

It seems

DSPG

GAG

calcification.

remain

immunocytochemical

served.

that

of PG core

who

there

in the present

staining

the concept

study

is presumably

mechanism

surrounding

tion

cartilage

observations

DSPG

im-

with

core

during

observations

small

monomer that

examined

DSPG

proteolysis

et al. (40),

postulating

proteins

these

the

ing

by Poole

D,

and

immunohistochemical

cartilage

cartilage,

supports

alter

in aqueous

specific

study.

as cathepsin

in GAG

in agreement

degra-

hypoth-

demonstrat-

such

decrease

matrix

not

the

immunostaining calcified

findings

accompanies

evidence from

plate

between

The

in the dentin

proteolytic

(5). This

enzymes,

(39).

or removal

vation

that

mineralization

of PG-degrading

munostaining

loss

it is believed

matrix

immunohistochemical

predentin

degradation

growth

the

with

presence

in the

and

initiates

antibodies

im-

staining of dentin cannot be ruled out. PG inhibit the growth of hydroxyapatite crystals (9,26) and the initial formation of calcium

demineralized

with

good as PG

munohistochemically those

strongly

small

deminer-

providing

materials

more

and

specimens

towards cationic sites. However, the possibility that a change in conformation of the molecules may explain the decrease in immuno-

for PG than

stain

except

noted

alkylammonium

matrix

the tissue during aldehyde and that this may result in

of the dentin

However,

in ethanolic in the

from

Calcif

Tissue

Int

41:52,

1987

7. Caterson B, Calabro T, Hampton A: Monoclonal antibodies as probes for elucidating proteoglycan structure and function. In Wight TN, Mecham RP, eds. Biology ofproteoglycans. Orlando, FL, Academic Press, 1987, 1 8. Caterson B, ChristnerJE, tibody that specifically Monoclonal antibodies 1983 9. Chen

CC,

proteoglycans

BakerJR:

corneal

to cartilage

proteoglycan.J

AL. Rosenberg on hydroxyapatite

Boskey

10.

Clark RD, aminoglycans

11.

Coster

L, Fransson

phate

proteoglycans

Identification

recognizes

Smith JG Jr, in human

ofa

and skeletal

Hexosamine Biophys

L-A Isolation and characterization from bovine sclera. Biochem

keratan

Biol Chem

LC: The inhibitory growth. CalcifTissue

Davidson EA: teeth. Biochim

monoclonal

an-

sulfate. 258:8848,

effect of cartilage mt 36:285, 1984 and acid glycosActa 101:267, 1965

ofdermatan

)

193:143,

sul1981

we could

in the

dentin,

of several

animal

12.

CouchmanJR,

Caterson

clonal antibody detection Nature 307:650, 1984

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

B, ChristnerJE,

BakerJR:

of glycosaminoglycans

Mapping

in connective

by mono-

tissue.

324

13.

TAKAGI,

Dziewiatkowski dral

DD,

ossification.

Majznerski

Calcif

Tissue

a:

Role ofproteoglycans

Int

37:560,

Embery G: The teeth of young 14:59, 1974

isolation ofchondnoitin rats receiving sodium

4-(355)sulphate (35S)sulphate.

15.

Engfeldt B, Hjerpe A: Glycosaminoglycans Calcif Tiss Res 10:152, 1972

ofdentine

16.

Fiore-Donno humaine.

from the molar Calcif Tiss Res

G, Baume Odont

U: Etude histochimique Acta 10:141, 1966

predentine.

de Ia dentinogenese 38.

18.

Goldberg M, Escaig F: Incorporation of (35S)sulfate and (3H)glucosamine into glycoaminoglycans in rat incisor predentine and dentine: comparison by autoradiography of fixation by rapid-freezing, freezesubstitution and aldehyde fixation. Calcif Tissue Int 37:511, 1985

M, Septier

D: Electron

cans in rat incisor predentine Oral Biol 28:79, 1983 21.

22.

23.

24.

reaction

Goldberg

M, Septier

microscopic

components

ruthenium

hcxamine

of proteogly-

with cuprolinic

blue.

ofproteoglycans

and

predentine

and dentine

in rat incisor trichloride.

Arch

Oral

Biol

31:205,

tin

A, Antonopoulos

CA,

glycosaminoglycans

phy.

Calcif

using

Tissue

Int

25.

Hjerpe

26.

cif Tiss Res 22:173, Howell DS, PitaJC, molecular inhibitor(s)

from

A, Engfeldt

calcifying

high-performance

35:496,

liquid

of dentine

and

of den-

predentine.

Cal-

Jones IL, Leaver sue Res 16:37,

29.

Karnovsky MJ: A formaldehyde-glutaraldehyde laity for use in electron microscopy.

31.

32.

Li CY,

Ziesmer

LF, Greulich

by radioautography.

J Cell

RC: Formation NY Acad

Ann

Lazcano-Villareal

0:

Use

as an inhibitor forendogenous peroxidase method. J Histochem Cytochem 35:1457,

33.

Linde A: Dynamic aspects of dentinogenesis. chemistry and biology ofmineralized tissues. Media, 1985, 344

34.

Linde A: Noncollagenous proteins sis. In Linde A, ed. Dentin and FL, CRC Press, 1984, 55

and

Linde

1989

A: Ultrastructural

B, Hansson

localization

region

H-A,

localization

Rahemtulla

Linde

ofrat

of pro-

incisor.

A: Cathepsin

in dentinogenesis.

45.

F, Butler

glycosaminoglycans

Cell Tis-

D: ultraim-

Calcif

Tissue

Int

WT

Incorporation

by mineralized

tissues

of(355) sulphate vivo. Biochern

in

J

1984

Rahemtulla F, Malinstr#{246}m A, Heineg#{226}rd D: Monoclonal antibodies dermatan sulfate proteoglycans of soft connective tissues. J Dent 67(spec iss):373a, 1988 Rahemtulla

F, Prince

CW,

of proteoglycans

Butler

from

M, Pras M: Ground

precipitation

of calcium

WT:

Isolation

rat incisors. substance

phosphate.

and

Orthop

to

Res

partial characterJ 218:877, 1984

Biochem

proteinpolysacchanides Clin

Kyffin 1W: Demineralization salts ofethylenediaminetetra-acetic

ScottJE.

monium 46. SmalleyJW,

47.

48.

and the

60:235,

1968

in organic solvents by alkylamacid. BiochemJ 169:697,

WT,

ed. The AL, Ebsco

1980

Sundstr#{246}m B: New

dentin

formation.

1.kagi

M, Parmley

aspects

on the utilization ofinorganic 26:61, 1971

RT, Denys

FR: Ultrastructural

in odontoblasts,

predentin,

50.

Tokuyasu

KT: Applications istry. 3 Microsc 143:139,

Vila-.Porcile

E, Picart

52.

H,

tissue:

Sacks

35:287, CR,

inhibition

of the

ofcryoultramicrotomy

of corn-

J Histochern

peritubular

rna-

to immunocytochern-

A, Tougard

of laminin

Cytochern

Weinstein

nective

localization

and dentin.

1986

R, Tixier-Vidal

distribution

Histochern

during

1981

l#{224}kurna5, Eda 5: Structure and development nix in dentin. J Dent Res 45:683, 1966

‘cellular

sulphate

Histochemie

49.

1961

odontogenesis. 23:328, 1970

Embery G: The influence of fluoride administration on of proteoglycans in the developing rat incisor. Biochem

plot carbohydrates Cytochern 29:747,

osmo-

of azide and hydrogen in the immunoperox1987

in adult

C: Cellular

rat anterior

and

sub-

pituitary.

J

1987

Schubert

M: Protein-polysaccharide

of phase

separation.

Science

142:1073,

in con1963

53. Weinstock A: Elaboration ofenamel and dentine matrix glycoproteins. In Bourne GH, ed. The biochemistry and physiology of bone, Vol 2. New York, Academic Press, 1972, 121 54. Wislocki GB, Sognnaes RF: Histochemical AmJ Anat 87:239, 1950

reactions

of normal

teeth.

55. Woodward C, Davidson EA: Structure function relationships of protein polysaccharide complexes. Specific iron binding properties. Proc NatI

proteoglycans

dentinogenesis.

ofhigh

of bones and teeth Sd 60:630, 1955

In Butler Birmingham,

1973

M: Autometalpolyanions with

1982

CW,

44. Schubert

CalcifTis-

Biol 27:137A,

Provenza DV: Mucopolysacchanide in and autoradiographic study. Histochemie SC,

92:249,

Prince

ization

51.

fixative

peroxide idase

dentine.

1974

CP, Belanger

Lennox DW, Histochemical

ofhuman

37:203,

12:281,

1979

the structure 3 190:263,

28.

Leblond

43.

chromatogra-

1976 MarquezJF, Gatter RA: Demonstration of macroofcalcification and nucleational factor(s) in fluid sites in cartilage. J Clin Invest 48:630, 1969

AG: Glycosaminoglycans

H-A,

H, Persliden

into

1983

B: Proteoglycans

DLE, Goldberg of rat incisor

Cytochem

layer in

Res

1978

B, Wikstr#{246}m B: Analysis

Engfeldt

Hunter GK, Grynpas MD, Cheng P-T, Pritzker KPH: Effect of glycosaminoglycans on calcium pyrophosphate crystal formation in collagen gels. Calcif Tissue Int 41:164, 1987

as visualized

H, Hansson

Nygren

Biol 41.

of injected kidney: ulCytochem

D, Denmat visualization

Tissue

40. Poole AR, Pidoux I, Rosenberg L: Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate. J Cell

42.

27.

30.

Nygren

29:251,

1986

Graham RCJr, K.arnovsky MJ: The early stages ofabsorption horseradish peroxidase in the proximal tubules of mouse trastructural cytochemistry by a new technique. 3 Histochem 14:291, 1966

Calcif

Nagai N, 1?akuma 5, Goto Y, Ogiwara H: Electron microscopy of dentine and predentine of developing rat molars stained with ruthenium red. J Biol Buccale 2:73, 1974

munohistochemical

using

Goldberg M, Triller M, Escaig F, Genotelle-Septier D, Weill R: D#{233}tection sur coupes ultrafines de mucopolysaccharides acides par Ic bleu alcian dans des tissues dentaires inclus en #{233}pon. Biol Buccale 4:155, 1976

Hjerpe

39.

Arch

membrane

teeth.

J Histochem

blue.

RAHEMTULLA

of the odontoblast-predentine

porcine

Lorm#{233}eP, L#{233}colle 5, Septier lography for histochemical

224:941,

D: Visualization

associated

visualization

and dentine

active

KAGAMI,

teoglycans in the odontoblast-predentin sue Res 168:277, 1976

in hu-

Goldberg M, Genotelle-Septier D, Weill R: Glycoprot#{233}ines en prot#{233}oglycans dans Ia matrice pr#{233}dentinaire et dentinaire chez Ic rat: une #{233}tude ultrastructurale. J Biol Buccale 6:75, 1978 Goldberg

36.

37.

Fullmer HM, Alpher N: Histochemical polysaccharide man developing teeth. Lab Invest 7:163, 1958

20.

A: Glycosaminoglycans

cuprolinic and

17.

19.

35. Linde

dentinogenically

14.

Helv

in endochon-

1985

HOSOKAWA,

HISHIKAWA,

Acad

Sci USA

in dentinogeneVol II. Boca Raton,

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on April 30, 2015

60:201,

1968