The relationship between dentin microhardness

and tubule density

Pashley D, Okabe A, Parham P. The relationship between dentin
microhardness and tubule detisity. Endod Dent 'Frautnatol
1985; 1: 176-179.
Abstract - The purpose of this study was to determine the possible
eorrelation between dentin microhardness and dentin tubule density in normal human permanent teeth. A new technique was
developed whieh permitted serial determinations of the tnierohardness and tubule density of the same group ol titbules, beginning tiear to the detititio-etiamel junctioti and progressitig to
the pul|i ehamber. I'lie results show that there is a highly statistically significant inverse eorrelation between dentin microhardness and tubular density. 'Fubular density increased as the
pulp ehamber was approached. Fliis was associated with a
decrease in dentin mierohardness, presutnably due to a decrease
in the amount of intertubular detitin atid an iticrease iti iticlividttal
tubular diameter.

Numerous invesligators have tneasurrd the hardness

of dentin (1). Several investigators have reported
that detitin tnierohardtiess varies iti different parts
of a given tooth (3, 4, 13). Craig et al. (3) reported
Knoop hardness numbers (KHN) of dentin that
ranged from 35 to 83 while Inisayama et al. (4)
reported a range of 20-70 KHN. 'Fhis wide variation is disturbing to those tryitig to tneasure
ehanges in microhardness befbre atid after experimental procedures (1, 12). Craig et al. (3) suggested that dentin microhardness tnight be correlated
with tubule density in view of the faet that dentin
hardness deereased as measuremetits were tnade closer to the pulp. iMjsayama et al. (4) also clearly
demonstrated that dentin KHN deerea.sed as mierohardness was measured from the dentino-enamel
junetion (DEJ) toward the pulp ehamber. A sitnilar
eorrelation can be made between dentinal tubular
density and distanee from the pulp ehamber (5).
No one has yet statistically tested the correlatioti
between microhardness of dentin and tubular density.
Material and methods
Preparation of teeth
Uncrupted human 3rd molars were utilized for this
study. 'Fhey eame from individuals of both sexes
176

David Pashley, Atsuko Okabe and

Philiip Parham
Departments of Oral Biology and Physiology, Medical College ol Georgia, Augusta, Georgia, USA

Key words: dentin rtiicrohatdness, tubule density.

D. Pashley, Department of Oral Biology, Medical
College of Georgia, Augusta, Georgia 30912, USA.
Accepted for publication 20 May 1985.

ratigittg in age from 17 26 years. After extraetioti

they wete stored in phosphate buffered saline (Dulbeeco's PBS) at 7C for periods ranging frotn 1 20
weeks.
Fhe roots were sectioned from the crown at the
level of the cemento-enatnel jutietion using a diatnotid saw (Isotnet, Buehler, Ltd.). The occlusal
enatnel was removed by a second seetion tnade parallel to the first to produee a crown segment as
previously described (9). After removing the pulpal
soft tissue with ati endodotitic broach, 0.1 \x\ of0.5%
trypati blue or cotigo red dye was placed in the ti]i of
the pul|5 horn using a 1 [xl tnicro|jipette (Microcaps,
Fisher Scientific Co.). The dye was allowed to diffuse to the dentin surface, thereby tnarkitig a group
of tubules from the pulp to the surfaee. Those teetli
that did not pertnit dye to difluse to the surface
were disearded. In some cases, the dye movetnent
was aeeelerated by plaeing the crown segment iti a
split ehamber device (9) and applying 10 psi of air
|)ressure to the pulpal side to foree the dye through
the dentin. By dyeing the teeth in this tnanner,
hardness measuretnents could be taketi on the same
groups of tubules throughout the experitnent (Fig.
1).
After the clye had cotnpletely dilfused through
the tubules, the dentin surfaces were polished. First,
the specimen was polished on 600 grade emery pa-

Dentin microhardness and tubule density

Pig. I. Sehematie representation of the manner in whieh the

crown segment was prepared and subseqnently studied in
sequential sliee.s. The stijipled areas indieate the dyed areas of
dentin in whieh niierohardne.ss measurements were made.

p e r to remove scratches created by the saw. The

specimen was then polished on a felt material (AB
Microcloth, Buehler, Ltd.) in a 1.0 fxm alumina
slurry. 'Fhe tooth was exatnined under a microscope,
a n d if scratches still remained it was again polished
i n the 1.0 (im slurry. 'Fhe specitnens were kept tnoist
throughout the experitnent by placing thetn in wet
chambers (botdes lined with wet ti.ssue paper). AH
specitnens were oriented perpetidicular to the ind en ten
Determination of microhardness

Mierohardtiess measuretnetits were only made

within the dyed zone of dentin (Fig. 1), using a
JCnoop indenter tnierohardtiess tester ('Fukoti Mierohardness Tester, Model MO) under a load of
lOO grams to tninimize tneasut-enient variance (11).
Indentation lengths were tneasured as Filar units
wliich were cotiverted to Knoop hardness nutnbers.
Indentations were made every 108 |im across the
d y e d portions of the dentin surface. Eaeh slice received 17 to 81 indentations, depending on the size
o F the dyed areas of detititi.

in cross section; tangentially-cut tttbitles were excluded.

Eaeh speeimeti was sliced a ntmiber of times,
approximately 0.5 to 1.0 tnm frotn the preeeditig
detitin surfaee (Fig. 1). Hardness measuretnents
wete repeated on the dyed dentin portion and
photographs were taken to calculate the tubular
densities. When the tips of the pulp hortis were eut
off, usttally on the fitial sliee, indetitations were
made next to the edge of the exposure, since the
tiumber of tubules was highest in this area. There
was no statistical difference between KHN made by
plaeitig the indenter jjarallel to, at right angles to,
or at any other angle to adjacent indetitatiotis.
Speeimen 1 was sliced a total of six times, specimens
2 and 3 ftve times, atid specitnens 4 atid 5 twice.
Fhe correlations between microhardness and tubule density were atialyzed using the ANOVA litiear
regression equation (Hewlett Packard, Model 9801
A).
Resuits
Data from 5 permanent, tttierupted 3rd tnolars are
showti in Fig. 3. Plotted on the ordinate are the
Knoop hardness tiumbers (KHN) of 216 observations in whieh tubular densities were also tneasttred. Fhe hardness \-alues ranged from 25 to 81.7,
while the tubttlar detisities ratiged from 1.95 lo
5.65 X 10' tubules mtii -. Ivicli of the 5 teeth is
represented by differenl symbols. Fhe linear regressioti eqitation itidicates that the KHN of detitin,
at zero tubular detisity would be 97.8, while the
tubular detisity at a KHN of zero (predentiti or
odotitoblastic layer) would be 8.29 x 10' tubules
mm '-. 'Fhe data show cotisiderable scatter but the

Determination of tubule tjensity

W i t h a metallograph (Unitron Metallograph, Model BNX-1 1), photographs were taken of the indentations under a magnification of 240 x . On the
photographic print, a 5 mm x 50 tnm rectangle was
drawti alotigside of the lotig axis of the indentation
yielding a knowti, eonstant surface area of 4340 |atn^
(Fig. 2). The number of tubules within the reetatigle
w a s counted and expressed as a tubular density
(number of tubules tiim^''). All tubular detisities
w e r e measured oti satnples whose tubules were cut

.;/:

Eig. 2. .Side A shows the tiibniar density and mieiohardness

indentation ofdentin sampled near the (lentinoenaniel jmu tion.
Side li (lemonstrales the lubular- density and mierohardne.w i)identation of dentin near the pulp ehamber. Both photographs
were taken al ihc .same magnilieation. The larger indentation
in H indicates the dentin is softer ihan that sampled in A.

177

Pashlev et al.
90-1

OSPECIMENI
SPECIMEN2
X SPECIMEN 3

ASPECIMEN4
a SPECIMEN 5

8070CO

aR

X 50-1

oo 5

40
y=-ijSxio^x-HS/s
30
20

o.,, ^^^

r = -O.bl
p i O:CX)I

6.0
3.0
4.0
5.0
DENTIN TUBULAR DENSITY x lo'* TUBULES tnm"^
Eig. 3. The inverse relation.ship between dentin Knoop hardness
number and tubular density is demonstrated by the regression
line. Eaeh symbol represents a dilTerenl (ooth whieh was sampled
at dilterent^ distanees from the, pulp. Note the high eorrelalicm
eoelTieient.
2:0

correlation coefficient ( 0.61) is highly statistically

significant (p<0.001). Individual teeth generally
gave a higher correlation between these two variables, as is shown in Fig. 4. In this figure, the
sequendal slices that were used to reduce dentin
thiekness were labelled A, B, C, D, E and F. 'Fhe
different symbols designate the data obtained from
the appropriate slice. 'Fhe highest Knoop hardness
numbers were usually obtained on the secotid slice
and the lowest numbers on the last slice (compare
Fig. 1 with Fig. 4).
Attempts to fit the data (o exponential regressions
or power functions gave lower correlation coefficients.
90i
.-^ 80
X

SLICE A
o SLICE B
X SLICE C

w 70-

A SLICE D
O SLICE E
e SLICE F

i
1 60
X

2 50
40
30

y = - l.37xlOx + IO86
r = -0.70
n=l27
t= 10.9
p< 0.001

3.0
4b

5'.0

6b
DENTIN TUBULAR DENSITY x lo''TUBULES tnm'^
/'/,<;. 4. I he relationship between dentin hardnes.s and tubular
density is shown in a single tooth as a funetion of sequential
sliees.

178

2.0

Discussien
The data shown in Fig. 3 represent, to our knowledge, the first time that mierohardness values have
been obtained on the same group of tubules at
different distances from the pulj) whieh were also
correlated with dentinal tubular density. 'Fhe highly
significant inverse eorrelation between KHN and
tubular density is striking. 'Fhe hardness values obtained in this study are similar to those previously
published by others (3, 4, 10), as are the tubular
densities (5). Garberoglio & Briitinstrotn (5) reported a tubular density of 45,000 tubules/mm'^ near
the pulp. 'Fhis is somewhat lower than the 82,900
tubules/mm''' projected by the regression equation
at zero KHN from Fig. 3 (i.e., in the odotitoblastic
cell layer). Ketterl (7), however, reported tubular
densities of 70,000 per mm'-' elose to the pulp, whieh
are mueh nearer to the value that we obtained.
Our data (Fig. 3) support and extetid the studies
oi'Craig's group (2, 3, 10), who demonstrated that
dentin is softer just beneath the DEJ relative to
deeper crown dentin. This was attributed to the
presence of interglobular dentin which is richer in
organic material than regular dentin, as well as to
the greater number of bratiehes in the tubules at
the DEJ. Craig et al. (3) also reported that dentin
beeame softer near the pulp because it was "tnore
porous" but they did not test their hyi^othcsis. 'Fhese
observations were later eotifirmed by Fusayama et
al. (4) in longitudinal sections of hutnan teeth. Their
microhardticss "profiles" clearly detnonstrate dentin
KHN of about 57 at the DEJ, rising to 70 about
0.5 tnm deeper, atid then falling, gradually at first
and then more steeply as the pulp chatnber was
approached, to values near 20. 'Fhe use of longitudinal sections pertnitted them to fbllow the same
group of tubules fioni the DEJ to the pulp but did
not pertnit quantitation of tubular density or its
eorrelation to dentin hardness. Fhey gave no explanation for their reported regional ehanges in detitin
microhardness. Our data (Fig. 4) show that the
KHN measured in the seeond slice were higher than
those measured in the first slice, which would have
been closer to the DEJ. These data are in agreement
with those of Craig et al. (3) and Fusayama et al.
(4). Craig et al. (3) reported that large dilferenees
in erown dentin tubule number were noted but that
no measurable difference in microhardness eould
be detected. Fhese measurements were made on
transverse sections of teeth, as in the present study,
but were made randomly. 'Fhey were not satnpling
the same group of tuliules from iDerijiheral dentin
all the way to the pulp, as was done in this investigation. It is important to include as tnany observations
as possible at the two extretnes ol" dentin, namely,
just below (0.5 0.7 mm) the DEJ and just above

Dentin microhardness and tuhule density

(0.1-0.3 mm) the pulp. 'Fhese microhardness studies
seem to eonfirm older quantitative tnicroradiographic observatiotis of Thewlis (14), whieh gave
essentially the same descrijjtioti of changes in detisity
in the various portions ofdentin.
Hodge & McKay (6) reported variations in hardness in dentin in different areas of the same tooth,
b u t their method used a microscratch test whieh
lacked sensitivity and resolution. Craig's group (3)
was the fitst to demonstrate the differences in microhardness of dentin using microquantitative techniques. Fhey stressed the faet that the mierohatdness of dentin "must be deseribed in tertns of the
location in dentin", but they did not ofler atiy data
correlating microhardness and tubule detisity. 'Fhe
results of the presetit experitiients extetid Craig's
concept. It would appear that dentiti tniet oliat dness
must be described in terms of detitin tubular density
which varies in location of dentin. This statetnent
m u s t be qualified to hold true fbr tiortiial, primary
dentin. Marked differences in dentin sttucturc, such
a s the areas at the Dl'vJ (2, 3) and dentinocemental
j u n c t i o n (10) may deviate from this generalization,
a s would cariotis detitin (4), sclerotic dentin, pnlp
stones, ete.
The inverse relationship between dentin hardness
a n d tubular density is probably due to the direct
relationship bctweeti hardness and the amount of
caleificd matrix per mm^ ofdentin (12). Thus, as
t u b u l a r den.sity increases near the pulp, the atea
occupied by open tubules increases (15) to as high
a s 2 2 % (range 9.4-41.8%). The tubules offer little
resistance to indentation relative to that of calcified
intertubular dentin (12). Not otily are the tubules
rnore dense near the pulp, they are also larger in
d i a m e t e r (5) and free of peritubular detititi (8). All
o f these factors probably contribute to the decreasi n g K H N as the pulp is ap|3roaclied, and may be
responsible for the inability of clinicians to determ i n e exaetly when the pttlp chatnber will be reache d duritig exeavation of dentiti. 'Fhe relative softness
o f dentiti near the pulp chatnber obviously facili-

tates etidodontic itistrutnentatioti. Sitnilar studies

should be done on root dctititi to detertnine the
relationship between dentin hardtiess and tubule
density.
Acknowledgetttent - This work was supported by
Grant DE 06427 from the National Institute of
Dental Research.

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179