Journal of Histochemistry & Cytochemistry http://jhc.sagepub.

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KINETICS OF ACID DEMINERALIZATION IN HISTOLOGIC TECHNIQUE

HENNING BIRKEDAL-HANSEN J Histochem Cytochem 1974 22: 434 DOI: 10.1177/22.6.434 The online version of this article can be found at: http://jhc.sagepub.com/content/22/6/434

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THE JOURNAL OF H ISTOCHEMISTRY ANI) Copyright 1974 by The Hislochemical

CYTOCHEMISTRY

Vol. Inc.

22,

No.

6, pp.

4:34-441,

1974

ocietv.

Pr,ntedint.S.A.

KINETICS

OF

ACID

DEMINERALIZATION
HENNING

IN
BIRKEDAL-HANSEN2 Royal Dental August

HISTOLOGIC

TECHNIQUE

Institute

for

Cariology

and Received

Endodontics,

College, 21, 1973

Copenhs:igen,

Denmark

for publication

The rate of progression of the demineralization front during acid demineralization is investigated for standardized elephants ivory dentine specimens. A theoretical model for the penetration based upon a combined diffusion with reaction process is set up. It is demonstrated that the results are in accordance with the proposed model. For specimens of certain geometrical shapes (semi-infinite or plane sheet) the distance penetrated by the front is directly proportional to the square root of the time.
Demineralization is frequently an inevitable following toward in the processes: the

(a)
specimen

diffusion

of acid

in the

step has effect

21-25), around

in been of

hard paid various

whereas the

tissue

histology.

Much

attention bath
of acid

surface: (b) matrix

diffusion decalcified

to the more decalcifving

only little laws

or less deleterious agents (3, 4, 9,

interest has the

organic

of the

14, part

of the
(c)

specimen
reaction front; products. series but with

toward
and (d) The of events

the
outward exact is

decalcification
at diffusion kinetic complicated and and as stated with treatthe

gathered front;

hydroxyapatite

kinetic

governing

process. decalcification

It an

has been important

of

emphasized role (3, 23,

decalcification

that
30) is

and

diffusion that
significantly of decaicth-

plays of reaction the ment of this

elaborate, simplifving below, the

progression influenced

if certain are can

approximations made, be accomplished

by the

ing
ti()n on lying

solution
experiments nonstandardized physicochemical

in

rate of diffusion the specimen,

have mostly been specimens. phenomena

assumptions treatment

but
whereby are

decalcificaperformed underobscured

a reasonable The reaction

tite sion is considered of acid slower matrix The

result. between
instantaneous. decalcified diffusion inthe than diffusion problem

acid

and
As organic of acid

hydroxyapathe diffumatrix in the is bath

by have the front has tion

parts

uncontrollable revealed forward and further proceeds

of the

variables. that demineralization movement as a diffuse been

X-ray

techniques proceeds as

much (5),

not

of a sharply delineated loss of mineral salts. that rate, demineralization

is considered then is one

rate-deterof diffusion of

It mining.

demonstrated with decreasing requiring Brain (3) complete parts. difficulties specimens. not been
deals study

decalcitlcaacid in the central furnished much than this longer sites

solution sites as are

the
capable by

liquid
with a of an

phase
finite reacting

of an
number with reaction. crystals matrix

organic
of fixed the

polymer,
reactive diffusing

specimens

time the

to

achieve of the the have

Thus this

instantaneous

Reactive

peripheral

explained

hydroxyapatite

and

acid-bind(mainly collawith different

a result penetrate however,

tively.

for hydrogen ions These observations, expressed

with the

ing groups in the organic to gen). Hence we can consider

reaction process from

thediffusion two slightly

quantitakinetics

the

progression

of the

demineralization

front.

angles of low),
the the

(approach either (A)

liquid diffusion different phase

A
as simple of coefficient reactions-acid the

and

B
diffusion specimen,

as

discussed acid of
governed

bein
by with

THEORETICAL

From zation This Association praktiske

ten).
2

a kinetic point of view, of histologic specimens

acid demineralitwo consists of the

D0 and

combined binding to

colla-

gen

(5)

and as Dr,,

acid diffusion

dissolution of
by the with

of hydroxyapatite acid in
effective only one

(B) project was supported by the Danish Dental -or matrix, )Fonden til st#{246}tte videnskabelige for og undersgeiser inden for tandlaegekunsefficient,
request Institute of for reprints Cariology to: Dr.

the

collagenous
diffusion reaction, co-

governed combined

Address Dental

H.

Birkedal-

hydroxyapatite served bined relation

434

dissolution. diffusion

Dr,.

thus

is for

the the

obcomThe the

Hansen,

Royal

College,

and Endodontics, Universitetsparken, 4 DK 2100

over-all diffusion between

coefficient

Copenhagen

0. Denmark.

and acid-binding D0 and D, depends

process. on

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KINETICS

OF

ACID

DEMINERALIZATION

435

kinetics been
The

of discussed
total

the

acid-binding elsewhere (5).

of acid

reaction,
used for

as

it

has

z .io2

amount

complete

20

decalcification
expressed as

of

the

specimen

(M)

can

be
16

M=M,+M5#{247}M0 where
diffusing the

(1) amount of free, is pro12

M,
equilibrium

is

the

equilibrium

acid and
reaction Mh

contained
amount is the

in the
of acid

specimen,
bound

M0
to

teins
the

amount
If with

of
we

acid
assume diffusion

needed
that

for
proceeds

hydroxyapatite

dissolution. associated

to
of

completion
diffusing

in

the
there has

presence
as will occurred in exist at the

of small
case a front all reactive

amounts
of acid
10 20 40 60 a/b 80

substance,

demineralization, which reaction

behind sites FIG. 1. Graph of the function

Z.expZ2)

.erfZ

and
all.

ahead
As all

of which no
reactive

reaction
sites have

has

occurred
already

at a/b.
reacted

behind
as simple

the

front,
diffusion

the of the
lost

penetration
in the liquid

of acid
phase

occursFor
of the

approach

B correspondingly
Xv,
=

2Z0\/D

(8)

decalcified bath
stance

part of
is continually

specimen C0,
at but the front

from
diffusing

an instanhave

infinite
sub-

concentration reaction.
of Crank Hill analogous (10),

and TrZRexp(Z52)erfZR
=

by

(M,

(9)

taneous
Treatments given (17) by and

problems Dankwerts (11),

been Values Hermans

of Z can (18) for is

be
any

obtained
specified

from
value

graphs a/b. of 1) within

of the The the study. a/b)

the above

function function

(19). Details

of from
the

the these
solution

solution

plotted

below

(Fig.

procedure
For the

can
semi-infinite

be

otained
medium

authors. range ends Thus are

alization

of values needed for the present if the actual constants (D0, D, and determined,
front

up

with:
Xm
=

the
can

progression
be calculated

of the
from

deminer-

2ZVDT

(2)

equations.

where

Z is

given

by
=

If a equation
a/b 6 and 8

<<

b, expansion 3 gives a/b =

to:

2Z2

of and

the hence

functions equations

in

7r-Zexp(Z2)erfZ
Xm

reduce

is the

movement
t the

of the
time,

front,D the
exp the

diffusion
exponential
=

coefficient,

function, fraction reacted according

erf the between acid during to approach

a/b
=

error function. a/b is concentrations of free decalcification A:

+

the and

( M5 \
2(M,

2M,D,t

M0

(10)

or
=

and

hence,

M,,)D,,t M0 that of and

can 8 and

(11)

M,/(M5 approach

M5)

(4)

Practically acid needed greater than combining

plerfor equations equations 6

this

means

if the (M0) free M5)

be 9. used This is

amount
much

of acid
of

and

according
a/b

to
=

B: M)/M5
2 and 3 (5) gives

(M,

for demineralization the amount with

10 and

acid the

or siminstead situation

collagen
and 7 and

(M,
11

Inserting

this

in

equations

approach

A:
Xv,
=

2ZA.Ii

(6)

usually

prevails

during

decalcification

due

to

the teeth. It

considerable is seen from

mineral equations

content

of

bones 11 that

and the

and .HZAexp(ZA2)erfZA
=

M,/(M5

M,)

(7)

2, 10 and

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436
progression directly of proportional the demineralization to the square

BIRKEDAL-

HANSEN

front root of

is

r0

is

the

radius

of

the

cylinder,

r the

distance
known, front p

the from the

the
relative
-

center
distance can be p

to

the

front.
penetrated by as /1-0

When is 0
the

time
So

(parabolic
far we have

law).
dealt only sheet, suspended with the semi-infiby

(r0

r)/r0

calculated 1

nite
two

medium.
parallel

For
planes

theplane
and

hounded
in diffusing

(14)

solution provided substance nearly

the above that the

in

The error equations are also valid, concentration of diffusing conditions

of the sheet is zero or so equations

committed will be
12-14 process.
MATERIALS

by
small

assuming
provided a/b

steady is small, the deminis small. to

state

zero

the center (10). As

are pplicable a as a/b

AND of

the

reaction
is great practically meet at the

rate
and a/b until center

of acid eralization
is the of Cylindrical
either

actually
METHODS elephants

dissolution of hydroxyapatite small this will he fulfilled

two decalcification fronts

specimens in diameter

ivory

dentine,

the
For more as

sheet.
the infinite and law cylinder, no can the simple be found treatment relation (10, 17. is complicated the parabolic

3,0 mm mg and 2)
of

and (group of height a turning and

10.0 1) (500 the

mm or lathe.The end mg

of 10.0 dry

length mm weight) of of

(110:3

dr were
group

weight) 4.0 made mm on

such diameter 19).

(group
In
surface

curved
surfaces

connection approximate

with

demineralization, of Hill

however, (19) a
acid

the

1 specimens

treatment

is in reason-

group

2 specimens

were

double

lacquered

as

previ-

able accordance If it is assumed

concentration

with the conditions that there is

distribution of free

ously described (5). The specimens were decalcified in prevailing. 1.0 and 0.1 N HC1 at room temperature (22#{176}C)in the steady state presence of great excess of decalcif)ing fluid, which
behind
was changed

daily concentration performed solutions on

or

every of with were to

2nd hydrogen and not avoid on

day

to ensure ions.

constant stirbottom

the

front,
O
-

Hill
(1
-

gives
0) ln 1
-

the
=

following
Dt r,2b
=

solution:
4aDt r,2b 12)

external tion ring. were

of

Decalcificamagnetic the with specimens the

was When placed the

without stirred, contact of the

gauze The

where
0= 1-r02 (13)

bath. was D4,

progression Agfa-Gevaert.

decalcification radiographs at a 28 times

front (Structurix

measured

standardized Belgium)

magnification

(Fig.

2).

X-ravs

were

taken

at

regular

FIG.

2.

Radiographs
respectively,

of decalcifying,
in 1.0 N

Decalcification,

mineralized

and

demineralized

parts

cylindrical ivory dentine HC1 for 4, 8, 24, 48, 72 of the specimens.

and

specimens with only radial 79 hr. Note sharp boundary

acid

penetration. between

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KINETICS
DISTANCE 1mm)

OF

ACID

DEMINERALIZATION

437
obtained
weight

The
until

residue
constant

after
is

ashing

referred

5.0

content. The weight difference and mineral content is supposed

and firmly bound water reported (resistant

in oven at 650#{176}C as to mineral between dry weight to be organic content

to drying dentine at 110#{176}C).

//
40

From
firmly

the
bound

relative
water

amount contents
were

of
for

organic
other

material
samples

to and
con-

3.0 / 20
//

(14),
firmly

the

absolute
water

of
estimated.

organic
The

material
water

bound

tent of determined

the

decalcified part of the in a previous investigation

RESULTS

specimens (5).

was

10 /
/

The during
2 4 SQUARE 6 ROOT 8 OF 10 TIME HOURS)

progression decalcification in Figures penetrated to the

with

of in

the

decalcification

front

shown

FIG.

3.

Progression

of

decalcification

front

during

decalcification in 1.0 N HCI as function of square of time. Rodlike ivory dentine specimens with
axial note
line

root only
lines

tance portional

1.0 and 0.1 N HC1 is 3-5. It is seen that the disby the front is directly prosquare root of the time for
linear diffusion (decakifica-

acid means
gives

penetration. and standard the value of

Points and deviations. the penetration

vertical The

slope coefficient

specimens detion through of the

mens

with

end radial

surfaces diffusion

only), deviate

whereas clearly

specifrom effect was and I.

(2ZDH).
DISTANCE mm)

the parabolic relationship (Fig. 5). The of stirring on the rate of demineralization negligible mineral
50

(Table content of
=

III). a/b: (5) acid

Analysis before, q is

of it the part of the

organic is assumed fractional of the of the specimen.

is summarized As where

in Table

that
40 10!. EDTA

Calculation M, and the

qC,,V C0 the

pore men of
30

volume bath

of the and V the

decalcified concentration volume

specibulk

DISTANCE 20

mm)

10 40 8 16 SQUARE 24 ROOT 32 OF 40 TIME

HOURS)

30

FIG. calcification

4.

Progression in 0.1

of

N HC1

of square root of time. mens with only axial the line gives the value

decalcified front during deand 10% EDTA as function Rodlike ivory dentine speciacid penetration. The slope of of the penetration coefficient

20

10

(2ZDH),
20 40 60 TIME 80 IHOURSI

intervals

until

decalcification

was

completed.

After

each
the mens room and after

examination,
decalcifying were decalcified

the
solution.

specimens
For in 10

were
comparison buffer

put

back
a few

ethvlenediaminetetraa(pH 7.0) for to as their dry

cetic acid (EDTA)

temperature. specimens content. at Further mineral drying

in 0.2 Tris M
were The 110#{176}C is

FIG. 5. Progression specidecalcification in Cylindrical ivory at acid penetration.

into

of decalcification front during 1.0 N HC1 as function of time. dentine specimens with only radial Points and vertical lines denote

means
analyzed constant referred organic curve obtained mined weight. tions weight

and standard deviations (-), theoretical (equations 12-14) for experimentally detervalue of D0a/b. - - - -, theoretical curve (equa12-14) normalized to the points.

Downloaded from jhc.sagepub.com by guest on September 10, 2011

438 Values viously here Mh acid moles unit in the

=

BIRKEDAL-HANSEN

of M, (5) conveniently

and and

M6 have are expressed

been

determined in per unit Table moles

pre- Ca,0(PO4)0(OH)2 II but In a strongly of of tionG proceeds: of volume.

8H lOCa5
+

-,

summarized p is the complete

(15)
+

6HPO4

2H,O the reac-

pG V, where needed for of

number of decalcification G

acid

medium,

however,

of hydroxyapatite volume of the I assuming phase; of The net the

contained specimen; pure hydroxyapatite for acids

Table mineral

within 1 is estimated and hydroxyapatite in from dissolution demineralization (3, 15, 18) is: Hence during tite.
equations

6H

-.

H2PO,,

(16)

andp is determined reaction in weak

the

H2PO4a total

6H

-,

H,P04 moles of

(pK

2)

(17)

stoichiometry process. of hydroxyapatite

of 20 values 5 are proposed term Xm/t

and

of

acid

is

needed

decalcification Calculated 4 and of 2 the

specimens

1 mole of hydroxyapaa/b of according to in model:

is constant the value 2ZDH.

shown

Table
for

II. to
plane

TABLE
Content of Ivory of
MmInitial Dry

I
Specimens per Unit

Check equation
sheet

According The so value B as (5) Z5 value

Dentin Volume

has

Wt

Wt

Organic era! I Material + Firmly Bound Content Water

mgig!

I
)

Or-

Hydroxyganic apatite Mate.rial#{176}

Equiva. lent
Amount of Acid (HCI)#{176}

parameters allow the 2ZD#{189} ither e and D91 and can are

and

constants

determined

far of

calculation by approach been in Figure in values. experimental Figure 5 by

of a theoretical A or approach determined Table 1 for

have

previously II and and ZA any specified values III, together

gmole/1 0.56 0.48 0.98

gmole 19.58

1.74
a

1,56

0.99

are summarized be read from Calculated summarized

Analysis Dental Calculated Calculated

provided

by

Biochemical

Department, Denmark. 5% water 15-17.

of a/b.

theoretical Table For radial results depicting

2ZDH of with

Royal

College, Copenhagen, by subtraction of according to equations

(13)).

(Eastoe experimental lated and strated in

diffusion, calcuare demondepth of pene-

TABLE
Parameters Acid (oncenN 1.0 0.1 Calculated M,/V M,/V rnole/g1 0.64 0.064 asa/b
=

II
from
15,

Calculated

Experimental Z4
.

Findings

I f
I
+

MR/V I
0.32

Z,

102

I
mm/hr

f
I
=

I
(M

19.58 19.58

I
0.32

32.2 3.2

49.0

19.6

12.6 3.6

15.6 9.8

I
3,9

2.2
0.67

M,/(M0
+

M)

according according

to approach to approach

A. B.

#{176}Calculated as a/b

Mb)/MR

TABLE
Calculated
Calculated from Equation

III
Values for Rate Xv,/t
Calculated from Equation 11

Theoretical 2z4D0#{176}
Calculated from Equation

and 2Z,,D,.,

Experimental

of

Decalcification

1
I
8

Xv,/t
Calculated from Equation_10],

I
I

Xv,/t
Experimental Stirring

Acid

(oncentrat

Xv,/t
Experimental + Stirring

ion

N 1.0 0.1 0.45 0.14 0.46 0.13 0.46 0.16

mm/h

r5 0.46 0.16 0.49 0.47 0.15 0.16 0.17 0.48

Values

obtained

from

two

different

samples.

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KINETICS

OF

ACID

DEMINERALIZATION

439 results penetration with (19). for the results who peneof

tion on time. Earls unpublished tration as a function oftime. Calculations using de Jong3 for frontlike equations 12-14 and inserting estimated values Bungenberg in cylindrical specimens accord of a/b and D0 gives the upper curve, predicting a obtained by Hermans (17) and Hill penetration rate somewhat faster than that observed. equations position
experimental

The

lower

curve

12-14 but was of the points.

observations

model, seem to

hut be

the

actual

proposed from the tration. tion that seen, that the is small (points) fit the Hill reacting values of either a/b or was calculated normalized It is with than expected.
assumption

an

approximate

model

Hills model is based upon the assumpthe amount of free diffusing substance compared to the amount of substance with
is

reactive
fulfilled

sites
during

in

the

medium.
decalcification,

This correobserved precylinder the between

somewhat
DISCUSSION

lower

and sponding predicted for

front models the meaning course of the of of penetration

the

results to Hills is 5). in the

root

obtained model. somewhat The the main plane

give although smaller difference sheet and

curve the than the

The and
rate validity of

agreement experimental
the of

between values
the made proposed in

theoretical the
supports despite of the the form of calcumodel of the

value
dicted

of aDo/b
(Fig.

penetration
the

demineralization

penetration is that the decreasing

square is a the later rate

approximations lations. The

rate for the

penetration

of penetration plane sheet

but decreases (Fig. 5). for

is continually according to
the initially cylinder, and

physical

relationship,

that
plane

for

specimens
sheet the

generally
depth

of increases

again

Hence time,

simple which (equaof hard enamel set

from

demineralization to the square tion coefficient, tional

cient specimen

front

is

directly time,
is directly

proportional with diffusion

part function of

calculation carried 2), cannot

of the

decalcification

is

root of the 2ZDH, which square

in further a/b phase the

a penetra- easily tion proporthe of

through for the be made for of refer 18).

differ as

plane sheet the cylinder. the only models kinetics to

to
of

the
acid and

root
is

of

the
a

decalcified

investigation coeffi- Previous tissue demineralization

16.

partition
in the liquid

factor

between
of the

the

amount

decalcified

dissolution (15, the these publications of acid present models, specimens

The be
organic disappears

up
the

in

considerably

might it
the

expected.
material during surface During

During
is decalis always neg-

and the amount of acid alizat ion reaction. For process. between square within directly concentration and 11). that
zation

needed for the the demineralizat

deminer- enamel ion ligible

cifIcation, in the contact tion that ganic is of

dissolution, and so with bone, matrix, generally that the

there the root certain

is almost penetration of this limits,

direct proportionality coefficient and

the bath.

mineral

demineralizathe w/w orof

factor, which means, the depth of penetration to the the

cementum which

and constitutes

dentine, 50-70

proportional From time

of a

of acid in equation needed for

sheet

the

plane

the tissue, is retained, so that acid can reach square root of the the surface only after diffusion in the bath (equations 10 mineral organic matrix of the specimen. In 2 it further appears, decalcified the present model the reaction between acid complete demineralihydroxyapatite is considered instantanespecimen is directly and

proportional the specimen.

to

the

square

of

the

thickness obeying the demonstrated

ous. According to Gray (15) the rate constant for of enamel dissolution in HC1 is approximately 50
>

A similar frontlike penetration so-called parabolic law has been experimentally tions
penetration previously cation For process. cylindrical specimens, the

10
this

g/cm2/sec.
investigation

Measured
(millimeter

in

the

terms
of

used

in

progression

in
into been

(1. 2, 6.7,

the front per square centimeter surface area per a great number of investigathis value corresponds to approximately 12, 20, 24, 27. 29) for fixative hour) 50 mm/hr. Compared with the actual penetratissue specimens. hut has it not ti()n rate for ivory dentine, mm 0.5 in the 1st hr. demonstrated for the decalcifiit frontlike appears reaction, is reasonable so rate-determining. de Jong Hermans HG: (17), that to assume diffusion It also an in follows instantanethe organic from results, modeous matrix no such can be
1926.

of
simple

penetration
relationship

is
as

still
the

present,
parabolic

but
law

Bungenberg

Unpublished

found

for

the

dependence

of

depth

of

penetra-

Cited by

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440 Grays investigation tion in weak acids, less (1/500-1/1000) rate so decalcification in strong, to bone can the dissolution instantaneous, only and The front close during involved changed cification. the small other seems than According concentration penetration N. Furthermore, teolytic cant. progress acid. to in to

BIRKEDAL-HANSEN

(15) that, for demineralizathe rate constants are and no of fully hence longer dentine, dissociated of the experiment obtainable 2 The and present the be model

ment the existence and direction of tubules and lamellae do not interfere with the rate of deminmuch eralization in different directions. Thus the rate reaction or of the front is not greater in the considered of penetration applies acids. direction of the tubules than perpendicular expected.
of acid

to This acids in be exob-

cementum decalcification probably penetration parameters 3 can hardly the rate of coefficient than for

the tubules as it might be probably is a result of the concentration used, served is elephants rate pected that arebe satisfactory decal-present when for any In as Sognnaes a lamellar ivory although
as model

rate of penetration in the present the acid in in greatest decalcification. equations order to CL the the

(28) using decalcification dentine. ivory So it

weak pattern might

in

dentine
substance

specimens
the useful

increase

Probably H and Further

diffusion ions is faster relative elephants most other

investigation, they studying demineralization

the present investigation,

maye b less in weak

the rate

acids.
of de-

mineral

content mineralization and

observation

is only
Similar

insignificantly
results were

be lower in dentine from to rate Brain will not beyond at this (3)

stirring. ivory dentine by Brain (3) species (13).

Clayden
that the

(8) of the

and
diffusion

influenced obtained by are consistent

of acid in

even

increase speed

of up of the

significantly a concentration concentration

with acid the the

the

decalcified by with Verdenius

of stirring that assumption

part stirring results and

was

specimen It is, however, by Molenaar The

by

is

not at (27)

influenced 1-2 variance pro-

(5). obtained Alma (30).

explained even

action of the acid will become Demineralization, therefore, usually with a penetration coefficient less This for the in
in of

signifi- and will effect less than the

lacking
Brain stirring

by (3)
in

vigorous

and frequently much histologic technique. acids and especially of penetration decalcification
demonstrated reaction,

than will

the bath 0.5 mm/hr in H inside the be true for weak For front
7.0 kinetics included has

will not specimen.

affect

the

liquid

contained

EDTA.

comparison, during
been of this in 1. Armstrong
tanning. Discuss

decalcification 10% EDTA pH

4. is The not

REFERENCES
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Soc

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processes

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Faraday

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model, specimens,

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decalcification

on unstandardized comparison with Further the

2. Baker JR: Cytological Technique. Methuen, Lonthe don, 1966, p 14-54 it appears that for plane 3. Brain EB: The Preparation of Decalcified Secparabolic relationship be- tions. Charles C Thomas. Springfield, Ill., 1966 EB, Eastoe JE: Studies in the decalcificaand time still holds. 4. Brain tion of dental tissues for histological purposes. Br (22, 26, 30) previous Dent J 112:277, 1962 have been per5. Birkedal-Hansen H: Diffusion of H36C1 in decalcispecimens, which fied ivory dentine. J Histochem Cytochem 22:428,

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access of acid 7. that the rate front devithis study.

8.

Burkl W, ide fixation.

Cafruny

Schiechl H: J Histochem
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that

under

the

conditions

of the

present

experi-

conduction

with

moving

boundary.

Trans

Fara-

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