Research

Remineralization across the resin-dentin interface:

In vivo evaiuation witli nanoindentation measurements,
EDS, and SEM
Naotake Akimoto, DMD, PhDVGen Yokoyama, DMD^/Kaoru Ohmori, DMD, PhDV
Shiro Suzuki, DDS, PhDVAtsushi Kohno, DDS, PhDVCharles F. Cox, DMD, FADI^

Objective; The purpose of this study was to evaluate the in vivo remineralization ol fhe possible non-tesin
infiltrated hybridoid layer between fhe hybrid layer and fhe subjaoenf dentin substrafe using nanoindenta-
fion, energy dispersive x-ray spectrosoopy microanalyses (EDS), and scanning elecfron microscopy (SEM)
technologies. Method and materials; Twenty Class V cavities were piaced in healthy adulf monkey teeth.
Each cavity was tofal etched with 37% phosphoric acid for 60 seconds, rinsed, and air dispersed, and SA-
Primer was applied to tfie collagen layer. Cavities were divided into two groups: In group 1. Protect Liner
(low-viscosity resin) and Clearfil AP-X (resin composite) were placed per manufacturer's directions, and no
bonding agent was placed on fhe acid-efcfied inferface. In group 2, Clearfil Phofobond (bonding agenf)
was applied, and Profecf LinerandClearfil AP-X were placed as in group I.Teeth were observed at 7
days (confrol] and 6 months by nanoindentafion, EDS, and SEM. Results: Six-monfh data sfiowed an
Increased nanchardness in areas 5 |jm adjacent fo fhe demineralized or partially demineralized denfin
inferface. Following treafment with a conventional adhesive sysfem on fhe acid-efched inferface (group 2].
fhere were increased nanohardness and calcium EDS measurements in the substrate just below the
resin-denfin impregnated layer. Conclusion: Our 6-monfh in vivo nanoindentation and EDS dafa demon-
strate fhaf the non-resin infiltrated zone becomes remineralized follcwing adhesive resin treatmenf.
(Quintessence Int 2001:32:561-570)

Key words: EDS observation, hybrid layer, hybridoid layer, nanohardness, nanoindentation, non-resin
impregnated layer, remineralizafion, resin-dentin inferface, resin diftusion, SEM observafion

emoval of the adhesion-inhibiting stnear layer

CLINICAL RELEVANCE: These in vivo dafa support tfie
ooncepf fhat remineralization occurs in tfie dentin sub-
strate beneafh fhe hybrid layer as a normal biologic
event, following a pattern of sclerosis seen under many
other resfcrative sysfems.
'Senior Instructor, Department of Operative Dentistry, Tsurumi University
School of Dental Medicine, Yokohama. Japan.
^Postgraduale Fellow, Departmenl of Operative Dentistry, Tsurumi
University Sctiooi cf Dental Medicine, Votoliama. Japan.
'Instructor. Department cf Operafive Dentistry, Tsurumi University Scfiool
of Dental Medicme, Yokohama, Japan
'Prcfesscr, Department ol Prosthodonlics and Bicm ate rials. School of
Dentistry. The University cf Alabama at Birmingham.
'Professor cf Dentistry and Chairman of Operative Dentistry, Tsurumi
University School ct Dental Medicine, Yokohama, Japan.
'Professor, Departments oi Comprehensive Care and Endodontics
ano Pulp Biclogy, School of Dentistry. The University of Alabama at
Birmingliam.
Reprint requests; Dr Naolake Akimoto, Senior Instructor, Department cf
Operative Dentistry, Tsurumi University School ol Dental tuledicine, 2-1-3
Tsurumi, Tsurumi-ku, Yokohama 230-6501, Japan. E-mail: akimoto-n@
tsL rumi-u.ac.jp
R with an acid etchant is now a widely accepted and
routine clinical procedure' especially to allow forma-
tion of a hybrid layer interface within the subjacent
dentin.2 Acid etching demineralizes the smear plugs,
the peritubular, and the intertubular dentin substrate.
Several studies have discussed factors that lead to for-
mation of the non-resin impregnated hybridoid
layer,^-' each factor being equally important for suc-
cessful long-term durability of the resin-dentin inter-
face- Variations in molecular weight of monomer sys-
tems may account for limited diffusive permeability
into the etched substrate. Other contrary factors are
an altered dentin substrate from aging, attrition, or
incipient or recurrent caries; sclerotic dentin; and
reparative dentin deposition. Perhaps most important
is the improper infiltration of the primer-resin system
into the demineralized substrate by the clinician, lead-
ing to formation of local or broad surface areas of
hybridoid zones, or incompletely filled substrate.
Consequently, this unfilled area beneath the hybrid
layer may remain hybridoid for some time. Kiyomura''

Quintessence internationai 561

 TABLE 1 Materials used in this study
Maferials Composifion
K-etc fiant 37% phosphoric acid
SA-Primer 3% 5-NlvlSA
80% etfianol in wafer
Phofobond
Cafalysf fulDP, HEMA, bis-GlvlA
Campfiorquinone
Benzoyi peroxide
tjniversai Aromatic sodium suiflnale
Tertiary aromatic amine in ethanol
Profecf Line Bis-Gh/A, TEG-DfViA
Microfilier (SiO^)
5-NMSA = N-metliaciyloyI E-aminasalicylic acifl; MDP = mettiacrylovioxy decyl diliydrogen ptiospliate; HEMA = 2-
hydroiyettiyi methacrylate; bis-GMA - bisphenol glycfdyl methacrylate;TEG-DMA = irielhylere glycol dimethacrylate;
SiOj = silicon dioxide.
and Takarada et aF have speculated that the hybridoid
layer may adversely affect the long-term bond stability.
However, contrary to a few in vitro bond tests that
tbeorize poor long-term bond durability, clinical per-
formance of the many adhesive composite restorative
systems available on today's commercial market con-
tinues to demonstrate promising results, regardless of
the hostile environment present in the mouth.'""'^
To date, few studies have evaluated long-term in
vivo changes across the resin-dentin interface.'^'^
Consequently, the authors' hypothesis is that in vivo
remineralization of the hybridoid layer will occur
over a 6-month period. The aim of tbis in vivo study
was to measure the nanohardness and Ca content
across the resin-dentin bond interface in monkey
teetb, employing nanoindentation and energy disper-
sive x-ray spectroscopy (EDS) technologies through
the resin-dentin bond interface of each measurement
site and its vicinity.
METHOD AND MATERIALS
Following university internal review board approval,
a 4-year-old adult rhesus monkey was conditioned
for 90 days in tbe animal facilities. The monkey pro-
vided 20 vital teeth for tbis study. Ali teetb were
scaled and polished with a rubber cup and prophy
paste prior to each operative sequence. The animal
was tranquilized with an intramuscular injection of
10 mg/kg of ketamine hydrochloride (100 mg/mL),
Muscular immobility was maintained witb an intra-
muscular injection of xylazine bydrochloride. Quad-
rants were isolated with sterile gauze and cotton
rolls. Twenty teeth received a Class V cavity through
the enamel Into the dentin with a 330 carbide bur,
copious water spray, adequate flusbing, and bigb-vol-
ume evacuation. A new bur was used on every fourth
562
Lof No. fvlanuiacfurer
160 Kuraray
045 Kuraray
Kuraray
256
360 Kuraray
0032 Kuraray
cavity to ensure superior cutting efficiency. The depth
of each cavity was approximately 1.5 to 2.0 mm. To
avoid vascular compromise of the dental pulp, no
local anesthetic was employed.
Table 1 lists all restorative materials used in this
study. Ten teetb were observed after 7 days, and 10
teeth were observed at 6 months postrestoration. Each
cavity was total etcbed with 37% H^PO^ K-etcbant for
60 seconds to over-etch the entire vital dentin sub-
strate. Tbe cavity was liberally rinsed with sterile water
and gently air dispersed for 5 seconds. SA-Primer was
applied for 10 seconds with a new plastic brush, and
the cavity walls were gently air-dried for 5 seconds.
Group 1 (n ^ 10) consisted of five cavities created at 7
days and five cavities created at 6 months. Each cavity
received an application of Protect Liner to the axial
floor and walls and was light cured for 40 seconds
with an Optilux 400 (Demetron}. The remainder of
the cavity was restored to the cavosurface margin with
Clearfil AP-X resin composite (Kuraray) and was light
cured per the manufacturer's directions. Group 2 (n =
10) received five cavities at 7 days and five cavities at
6 months. Each cavity was lined with Photobond
adhesive for 30 seconds using a new brusb for each
application, gently air dispersed, and light cured for 30
seconds. A thin layer of Protect Liner was then placed
on the axial floor and light cured for 40 seconds, and
the cavity was restored to the cavosurface margin with
Clearfil AP-X resin composite and light cured per the
manufacturer's directions.
The animal was sacrificed using left ventricular
flushing'^ with a 0.9% physiologic saline to avoid
introduction of fixative into the tooth substrates, Eacb
tooth was immediately cut from its alveolus without
placing any external traumatic stress to prevent distor-
tion or deformation of tooth tissues. Each tooth was
immediately placed into a 0.2''/o sodium azide isotonic
saline solution and stored at 4°C.
Volume 32, Number 7, 2001

Fig 1 Location ot nanohardness measurements at live specific
areas across the resin-dentin bonded zone ol ttie cavity (left) one
area on ttie occlusal cavity wall, one area on Ihe gingival cauity
wall, and three evenly spaced areas across the cauity floor.
Approximate nanoindentation Imprints made at points approxi-
mateiy 50 Mm from each ol the live indentation areas (right). PL =
Protect Liner; D = dentin.
Fig 2 Measured nanoindentalion points of group 1 (left) and group 2 (rigttt) A smali, pyramidal
impnnt is created by the uniform appiioation of a 100-mgi load per imprint tesl. PL = Protect Liner;
DD - demineralized dentin; D = dentin; B = Photobond; RiL = re s m-impregnated layer.
Nanohardness measurements
For preparation of tbe nanoindentation samples, eacb
of tbe 20 teetb was cross sectioned perpendicuiar to
the resin-dentin interface tbrough the center of
the cavity restoration using an MC-201 microcutter
(Maruto). Twenty sectioned halves of teeth from
groups 1 and 2 were embedded in impression tray
compound (GC) to stabilize each specimen parallel to
the stage of the nanoindentation device. Each surface
was individually hand polisbed with consecutive 800-,
1,000-, 1,200-, and 1,500-grit silicon carbide paper
(Marumoto) with tap water, followed by a diamond
paste of 6-, 3-, 1-, and 0.25-pm grit (Struers). After
polisbing, each ground sample was sonicated in dis-
tilled water using a Quantrex 140 ultrasonic device (L
& R Manufacturing) for 30 seconds to remove all
Quintessence International
• Akimoto et al
Enamel Dentin
debris. Eacb specimen was rinsed, allowed to dry, and
then flxed to its holder. Nanoindentation imprints
were made across the resin-denfln bond interface with
an ENT-1100 Nano Indentation Tester (Elionix).
Nanohardness was measured at flve specific areas
(Fig 1) across the resin-dentin bonded interface of the
cavity, one area on tbe occlusal cavity wall, one area
on tbe gingivai cavity wall, and tbree evenly spaced
areas along the cavity floor. Additional nanoindenta-
tion imprints were made at points approximately 50
pm from eacb of tbe five indentation areas. Figure 2
sbows representative indentation imprints for groups 1
and 2. A pyramidal nanoindentation imprint was made
by tbe uniform application of 100 mgf load per test.
Scanning electron microscopy (SEM) observations
were made of eacb indentation imprint using an ERA-
8000 electron probe surface-rcugbness analyzer
563
• Akimolo et al

140
1 |7d
120
*
1
Ë

"i SO

20

1
1 1
1
0
H
Demineraiized
1 1
Demireraiized
denlin + 5 [Jm
1 •_
Dentin
Protect
Liner
Photobond Resin- Resin-
impregnated impregnaled
Denlin

layer layer + 5 j m
aentir

Fig 3 Comparison of nanohardness oí the resin-dentin interface Fig 4 Nanoha'dness ol the resin-dentin bonded area in group 2
in group 1. ' = statistically signifioant at P < .01 by ANOVA and • = statistically significant at P < .01 by ANOVA and Fisher's PLSD
Fisher's PLSD test tor comparisons between hardness at 7 days test for ccmpariscns between hardness al 7 days and 6 months; n
and 6 rrionths, n = 10.

(Elionix). This instrument has four-channel secondary
electron detectors. The mode information generated
from the electron probe incident point on the speci-
men surface was detected simultaneously with four-
channel SE detectors. The electron probe incident
angle was found through processing data derived from
the intensity of the output signal generated from each
detector. In this manner, all complex surface morphol-
ogy was precisely measured. This instrumentation con-
tains topography, compositional, and secondary elec-
tron imaging (SEI) types. The data were statistically
evaluated by one-way analysis of variance (ANOVA)
and Fisher's protected least significant difference
(PLSD) test at P<.01.
application of a constant breaking force at two com-
mon points against a fulcrum. These specimens were
dehydrated through ascending grades of ethanol and
freeze dried in tertiary butyl alcohol in an ID-2 device
(Eiko Engineering). Each specimen was sputter coated
with tungsten in a high-resolution ESC-lOl-SEM
Superfine Coater apparatus (Elionix). Detailed SEM
analysis was made with a field emission electron probe
surface-roughness analyzer, ERA-SOOOFE {Elionix).
RESULTS
Nanohardness measurements

EDS microanalyses Figure 3 shows the results of group 1 at 7 days and 6

Two of five specimens from each group were îine ana-
lyzed by EDS. The same area of each specimen was
subjected to EDS to measure the Ca content across
the previously measured nanoindentation surface
areas using an ERA-8000 with EDX spectrometer
(Elionix) at an accelerating voltage of 15 kV and a
beam current of 5 X 10"' A.
SEM observations across the
resin-dentin interface
The remaining 20 cross-sectioned halves from the teeth
of groups 1 and 2 were each split longitudinally hy
months. There were no statistically significant difíer-
ences between the nanohardness of the demineralized
dentin (43.9 ± 15.5 mgf/ym^) and 5pm toward the dem-
ineralized denfin (52,7 ± 18.8 mgf/pm^) at 7 days. At 6
motiths, nanohardness of demineralized dentin showed
no significant differences, from 43.9 ± 15.5 mgf/pm^ to
45.6 ± 8.3 mgf/pm^; however, nanohardness measure-
ments 5 pm toward the demineralized dentin showed
significantly increased hardness readings from 52.7 ±
18.8 mgf/pmMo 83.1 ± 19.4mgf/nm2 (P<,01).
Figure 4 shows data of group 2 at 7 days and ö
months. Measurements of 7-day samples indicated
no statistical nanohardness differences between the
Protect Liner, 34.8 + 7.0 mgf/jim^, and Photobond,

564 Volume 32, Number 7, 2001


Fig 5 SEM view íiom group 2 at 7 days snows nano in dentation
imprints with a ioad ol 100 mgf onto the resin-dentin interface. The
composition of the area witnin 5 \im Irom Ihe border of the resin-
impregnaled layer (RIL] appears simiiar to normal sound dentin
(D). (Originai magnification X4,000.)
41.2 ± 11.2 rngf/jim^; and between the resin impreg-
tiated layer, 58.6 ± 16.0 mgf/iim^, and 5 pm toward
the resin impregnated layer, 53.8 ± 18.8 mgf/[im^.
Nanohardness measurements at 6 months signifieantly
increased in hardness 5 ym toward the demineraiized
dentin, from recordings of 53.8 + 18,8 mgf/pm^ to lev-
els of 81-7 ± 25.4 mgf/pm^ (P < ,01). Figures 5 and 6
are representative SEM views from group 2 at 7 days
and 6 months, respectively. SEM data showed nanoin-
dentations with a load of 100 mgf onto the resin-
dentin interface.
EDS microanalysis
Figures 7 and 8 are SEM views of compositional con-
trast SEIs, called compositional SEI (CSI), through
the Protect Liner, resin-impregnated layer, hybridoid
layer, and the dentin of group 2. Observatiort of den-
sity across the field of the CSI at 7 days showed a con-
stant density across the Protect Liner as a thin hori-
zontal line. The resin-impregnated layer presented a
different density than the hyhridoid layer, which
blended into a whitish zone. Upon characterization of
stirface topography, the hyhridoid layer presented con-
vex features that blended into a whitish horizontal
zone that terminated abruptly at the normal dentin.
Normal dentin showed a different density than the
other zones. A CSI at ö months through the Protect
Liner layer showed a lighter gray zone than the
darker-stained Photobond zone. A white line demar-
Quintessence internationai
- • • - ^ • • " " ' " " ^ . ' " I . . —
• AKimotc et ai
Fig 6 SEM view from group 2 at 6 months snows nanoindenta-
tion imprints (arrows) with a toad ol 100 mgf onto the resin-dentin
interlaoe. The composition ol the area within 5 ijm from
the border ol the resin-impregnated iayer (RiL) appears similar to
sound dentin (D] PL = Protect Liner; B = Photobond. (Oiiginai
magnitioation X4.000 ]
cated the Photobond from the slightly darker resin-
impregnated layer. There was a qualitative difference
in density from the resin-impregnated layer into the
hybridoid layer and below. The dentinal tubules also
appeared darker, similar to the hybridized resin zones.
An EDS scan of the Ca content across the resin-
dentin interface at 7 days (Fig 7) showed a level hori-
zontal reading through the Protect Liner resin and
through the resin-impregnated layer. As the EDS scan
reached the hybridoid layer, the Ca levels gradually
increased to a level similar to normal dentin. An EDS
scan (Fig 8) of the Ca content across the resin-dentin
interface of the Fig 6 specimen at 6 months showed a
continuous reading through the Protect Liner-
Photobond resin layer and resin-impregnated layers.
Once the horizontal EDS scan reached the termina-
tion of the resin-impregnated layer, considered hyhri-
doid, the EDS measurement of Ca rapidly increased to
a level equivalent to normal dentin. The 6-month in
vivo data show Ca levels in the hybridoid area within
5 pm from the border of demineraiized dentin at the
level of normal dentin.
Morphoiogic SEM observations
of bonding interface
Figure 9 shows field emission SEMs oí the split speci-
mens from group 1 at 7 days. An exposed collagen fib-
ril network approximately 5 pm in width was observed
between Protect Liner and sound dentin. Because
565
-
• Akimoto et al
Fig 7 SEM of a CSI shows the Protect Liner (PL], resin-jmpreg-
nated iayer (RIL), the hybridoid iayer (HOL¡, and dentin (D] in
group 2 at 7 days. Otiservation of density across the field shows a
constant density CSi gradient across the PL. The RiL shows a dif-
ferent density gradient than the HOL. Surface topography oharac-
terization ot the HOL presents conve*; features that blend into a
whitish horizontal ¿one that terminates abruptly at the normal
dentin. Normai dentin shovjs a different density than the other
zones An EDS scan of the Ca content aoross the resin-dentin
interface shows a ievei horizontai reading through the PL resin
and through the RiL As 1he EDS scan reaches Ihe HOL layer,
there is an increased Ca ievel ftiat reaches a lairly constant mea-
surement simiiar to normal dentin. (Ofiginai magnification
x4,000.)
Fig 9a Field emission SEM of split specimens of group f after 7
days. Exposed coiiagen iibrif zone approximately 5 [jm in width is
shown between Piotecf Liner (PL) and sound dentirt (D]. A gap is
seen between the PL and demineraüzed dentin (DD). presumabiy
because group 1 did not receive adtiesiue agenfs. The DD layer
shows a compiefeiy different stfucture fhan sound dentin.
(Original magnification x4,000.)
566
Fig 8 CSi at 6 months through the Protect Liner (PL] iayer. which
has a ijghter gray zone than the darker Photobond (B] zone. A
winife iine demarcates the B trom a dari<er resin-imp re g nated iayer
(RIL) zone. There is a quaiiLative density difference from the RiL
into the hybridoid layer and below. Note that the dentinai tubuies
aiso show a darker densify, similar to the hybridized resin zone.
An EDS scan at 6 months shows a ievei horizontai reading
through the PL resin, the B resin iayer, and the RIL. The horizontal
EDS scan reaches the ferminafion of the RiL into the hybridoid
iayer, and the Ca EDS measurement rapidly rises upward to a
ievel consistent wifh normai dentin (0). (Originai magnificafion
X4.000.)
Fig 9b Higher magnification of Fig 9a. PL = Protect Liner, DD =
demineraiiïed dentin (Originai magnification X1O,OOO.)
Volume 32, Number 7, 2001

Fig 10a Fieid emission SEM of spiit specimens of group i after 6
monlfis. The demineraiized dentin (DD) layer within 5 Mm from file
interface shows ooiiagen fibrils oniy in the topmost layer (black
star) and dense structures below (white star) Protect Liner is not
seen because adhesive agents were nol appiied. D - dentin.
(Originai magnification X4,000 ¡
Fig 11a Field emission SEM of spiit surfaces of group 2 at 7
days. Tfiree layers are seen between Protect Liner ¡PL) and sound
denlin (D¡. Adjacent to PL on the ieft side is a dark layer, presum-
ably Pfiotobond (B|, followed by a resin-impregnated layer, where
coilagen and 8 are present in mixture, and the hybridoid layer
(HOL) of 5 (jm wide, where apatite is removed and coilagen is
exposed. (Originai magnification X4,000 ¡
Ojinfessence infern at lonai
• Akimofo et al
Fig lOb Magnified view of Fig 10a leveais the presence of crys-
taiiized minerai (while star) DD = demineraiized dentin; black star
= coliagen fibrils oniy in the topmost layer. (Originai magniiication
x f 0,000.}
Fig l i b Magnifiea photo of Fig f f a shows resm-impregnated
layer (RiL), where Pliolobond (B) and coilagen are both present,
and a hybridoid iayer (iHOL), where apatife iias been washed
away and coilagen is exposed. (Original magnification x 10,000.)
567
•Akimoto et al
Fig 12a Field emission SEM photo in 6-month specimen shows a
Photobond (B) iayer adjacent to the Protect Liner (PL), wiiere
microtiliers ol ttie PL are recognized with the smooth B surlace,
and the res in-impregnated layer (RIL) layer, where B and collagen
are both presen!. Further, between the RIL and sound dentin (D),
torn ooliagen fibriis are seen. (Original magnification x4,000 1
group 1 did not receive an adbesive agent, gap forma-
tion was seen between tbe demineralized dentin and
Protect Liner. Tbe demineralized dentin showed a
completely different structure tban sound dentin.
However, at 6 months (Fig 10), the demineralized
dentin 5 pm from the interface showed collagen flbrils
in the topmost layer, with a more dense substrate
underneath. Again, a gap was seen between tbe dem-
ineralized dentin and the Protect Liner. Figure 10b is a
a magnified pboto revealing the presence of crystal-
lized mineral. Figure 11 shows field emission SEM
photos of split surfaces of group 2 at 7 days. Three lay-
ers were observed between Protect Liner and sound
dentin. Adjacent to Protect Liner was a darker layer,
presumably Pbotobond, followed by tbe resin-impreg-
nated layer, where collagen and Photobond were pre-
sent, and a 5-jim-wide hybridoid layer wbere apatite
was removed and collagen was exposed. Figure lib is
magnified photo of Fig 11a that shows the resin-
impregnated iayer where Photobond and collagen
were present and tbe hybridoid layer wbere apatite
was removed and the coiiagen was exposed. Field
emission SEM photos at 6 months (Fig 12) sbowed a
smootb Fbotobond layer adjacent to tbe tnore granu-
lar Protect Liner and a resin-impregnated layer wbere
Pbotobond and collagen were botb present. Split and
torn collagen fibrils were seen between tbe resin-
impregnated layer and sound dentin. Figure 12b is a
magnified view of Fig 12a; a dense area was seen
568
Fig 12b Magnification of Fig 12a. Dense structures are observed
within liie 5-|jm zone, wiiere interfibriiiar spaces are filied with
some precipilation (white star). Tiiis is the area where coiiagen fib-
rils were torn apart. This dense structure is quite different Irom the
one seen in Fig I t , where coiiagen fibrils were exposed. B =
PholObond (Original magnilication xtO.OOO.)
within 5 pm, where interfihrillar spaces appeared to be
filied witb some precipitaflon. Tbis is the area wbere
torn collagen fibrils were seen. The dense structure
was quite different from the one seen in Fig 11, where
collagen fibrils were exposed and thinly present.
DISCUSSION
Iiiyomura^ reported decreased in vitro bond strengtbs
from 17.5 to 3.7 MPa after 5 years, raising concerns of
adbesive failures in tbe deeper hybridoid layer.
Harasbima et aP later speculated that to ensure long-
term success, it is important to develop a homogenous
adhesive interface through the entire demineralized
dentin substrate. To study possible explanations,
Takarada et aP reduced the time of 10-3 acid etching;
however, they reported no change in bond strengtb over
time. This may have been because of the stabilizing
component of tbe ferric cbloride in the 10-3 etchant.
On the other hand, proper primer placement may have
promoted optimum primer inflltration into the etched
dentin substrate. Sugizaki" reported that the N-
methacryloyl 5-aminosalicylic acid primer system (SA-
Primer) opened the collagen fibril systetn, allowing for
tbe expansion of the coilapsed collagen network almost
to its original level. It was postulated tbat SA-Primer
would promote an ideal penetration of the adbesive sys-
tem into tbe demineralized dentin substrate.
Volume 32, Number 7, 2ÛOt

Nanoindentation tests on the resin-dentin interface
have evaluated elasticity and hardness," concluding
that the resin-dentin interdiffusion zone of collageti
fibrils is densely infiltrated with resin and that it cre-
ates an inherent elastic buffering mechanism that
compensates for polymerization contraction forces
within the resin composite. Those authors speculated
that an increased elasficity gradient (Young's modulus)
results from the highly elastic collagen meshwork and
the interdiffused resin. This gradient in stiffness occurs
between the adhesive resin and low-viscosity resin to
the rigid restorative resin, thus absorbing polymeriza-
tion shrinkage and stress generated by the resin com-
posite. Another group measured the hardness of the
bonding interface area including the resin-impreg-
nated layer at close intervals with a nanohardness-
measuring apparatus.'* They reported that the nano-
hardness of the non-resin impregnated hybridoid
dentin just beneath the resin-impregnated layer is
approximately 60% of that of sound dentin. SEM
analysis confirmed that the underlying dentin is a
non-resin infiltrated zone of demineralized dentin.
Consequently, long-term bond strength may likely
deteriorate unless remineraiization occurs within the
resin-free hybridoid demineralized segment.
To test possible changes in the non-resin impreg-
nated hyhridoid layer, we wanted to observe if etched
and demineralized dentin would remineralizo in vivo.
We also wanted to test if an adhesive system might
affect remineraiization of the demineralized dentin. In
our group 1 data, where only SA-Primer was used,
equal degrees of nanohardness were obtained in de-
mineralized dentin and areas 5 pm adjacent to the
demineralized dentin. Therefore, the use of SA-Primer
did not eliminate the hybridoid zone in deeper areas
of demineralized dentin. However, the nanohardness
within 5 pm from the border of the demineralized
dentin had significantly increased at 6 months com-
pared to our 7-day in vivo specimens. In group 2,
there were no statistically significant differences in
nanohardness between Protect Liner and Photobond,
or between the resin-impregnated layer and the hybri-
doid layer within 5 pm from the border of the resin-
impregnated layer. On the other hand, the nanohard-
ness of the hybridoid layer significantly increased at 6
months. The composifional SEI of 7-day specimens
(Fig 5) showed that areas within 5 pm from the border
of the resin-impregnated layer were similar to sound
dentin. We feel that this may be hecause of the pres-
ence of mineral. Nanohardness of this area was lower
than in sound dentin, as an increased degree of de-
mineralizafion had occurred from prolonged 60-sec-
ond acid etching. In the Fig 6 six-month specimen, the
area 5 pm from the border of the resin-impregnated
layer appeared similar to dentin, as in Fig 5. However,
Quintessence International
• Akimoto et al
the nanohardness of this area was higher than in 7-
day specimens. While the nanohardness of the
non-resin impregnated layer was slightly less at 6
months than sound denfin, the nanohardness of this
area showed a statistically significant (P < .01)
increase in Ca. EDS data (Figs 7 and 8) suggest that
the hybridoid layer adjacent to the resin-impregnated
iayer will remineralize after 6 months. These data
showed that remineraiization occurred in the hybri-
doid zone where the resin may not have thoroughly
penetrated. It is also suggested by field emission SEM
data (Figs 9 to 12) that 37% HjPO^-etched denfin will
remineraiize after 6 months, and that the hybridoid
layer in the interface of the resin-impregnated layer
will remineralize after 6 months without any influence
from the adhesive system. Consequently, our data
strongly suggest that an even partially acid-etched
demineralized dentin has the ability to remineralize to
nearly normal in vivo levels.
Numerous studies conducted on demineralization
and remineralizafion indicate that enamel and dentin
possess a capability to remineralize that is greater than
that of bone and equal to the capability of crystal
development of hydroxyapatite." Previous studies^""^^
have shown that the Golgi complex of odontoblasts
and other cells produces saccules that elongate and
give rise to small, abacus-like structures that contain
Ca matrix particles as well as collagen precursor ele-
ments. The Ca granules are known to be involved in
the normal physiologic minerahzation mechanism of
dentin. Bevelander and Nakahara^' demonstrated by
transmission electron microscopy (TEM) in rat pulps
that the onset of dentin mineralization occurs in
patches of acid mucopolysaccharide and on collagen
fibers within the odontoblasts, the mucopolysaccha-
ride being a prerequisite for dentin mineralization.
Reith^^ later reported that the major mineralization
occurs outside of the odontobiast at a later time; it
occurs along the predentin interface, taking part in
mineralization within the dentinai tubule adjacent to
the odontobiast process.
Using monkeys, Tatsumi^* demonstrated in vivo
remineralizafion in the zone of inner carious dentin,
suggesting that the physiologic remineraiization of the
inner demineralized zone occurs by normal biologic
processes. In that study, an adhesive system was
placed on the outer demineralized dentin. Therefore, it
is uncertain if an inner demineralized zone of dentin
may remineralize when resin hybridization is devel-
oped. Our in vivo data demonstrate a dynamic dimen-
sion to the study of biologic remineraiization in a
reproducible environment that is not possible in vitro.
In this study, vital dentin was demineralized with
H,PO, for 60 seconds, suggesting that even though the
hybridoid layer may be considered an obstacle for long
569
• Akimolo et ai
term bonding success, it has the biologic capability to
remineralize in vivo. Future studies from our group are
usitig TEM atid SEM to evaluate the in vivo pattern
and accumulation of Ca particles in odontoblasts and
their processes underneath various adhesively restored
teeth in hoth carious and noncanous nonhuman pri-
mate and human teeth.
Our 6-month in vivo nanoindentation, EDS, and
SEM data demonstrate that the non-resin infiltrated
hybridoid zone just beneath the resin-impregnated layer
beeomes harder following adhesive resin treatment.
ACKNOWLEDGMENTS
This investigation was supported in pan by grant-iii-aid No,
10771065 for Scientific Research from the Ministry of Education,
Science. Sports and Culture, Japan and UAB School of Dentistry
Restorative Research Grant No. 634717. In addition, the authors wish
to acknowledge the escellent technical assistance of Ms Michiyo
Yoshida and Mr Takuya Uemaisii, Mr Yoshio Taguchi, and Mr Hideo
Suzuki of Elionix, Tokyo. Without their personal support, none of
these data could have been generated.
REFERENCES
1. Fusayama T. New Concepts in Operative Dentistry. Tokyo:
Quintessence, 1980.
2 Naitabayashi N. Resin reinforced dentin due to infiltration
of monomers into the dentin at the adhesive interface. J Jpn
Dent Mater 1982;1:78-81.
3. Harashima 1, Hirasawa T, Totnioiia K, Okada J. Fracto-
graphy of the bonding between light-cured resin and tooth
substrates. Dent Mater J 1988;7:151-159.
4. Sugizalii J. The effect of various primers on the dentin adhe-
sion of resin composites-SEM and TEM observations of
the resin impregnated layer and adhesion protnoting effect
of the primers. Jpn ] Conserv Dent 1991;54:228-265.
5. Van Meerbeek B, Dhem A, Goret-Nicaise M, Braem M,
Lambrechts P, Vanherle G, Comparative SEM and TEM
examination of the ultra structure of the resin-dentin inter-
diffusion zone. J Dent Res 1993;72:495-50I.
6. Sano H, Takatsu T, Ciucehi B, Horner JA, Matthews WG,
Pashley DH. Nanoleaiiage: Leakage within the hybrid iayer.
Oper Dent 1995;20a8-25.
7 Tay FR, Gwinnett AJ, Pang KM, Wei SHY. Resin perme-
ation into acid-conditioned, moist, and dry dentin: A para-
digm using water-free adhesive primers. J Dent Res 1996,75:
1034-1044.
8. Kiyomura M. Bonding strength to bovine dentin with 4-
META/MMA-TBB resin-Long tertrt stability and influence
of water. J Jpn Dent Mater 1987;6:860-872.
9. Taitarada K, Kojima M, Ishihara K, Naitabayashi N.
Durabiiity of bonding between 4-META/MMA-TBB resin to
dentin pretreated with 10-3: The effect of 10-3 pretreating
period and subsequent glutaraidehyde treatment. J Jpn Dent
Mater 1990:9:831-840.
10. Shintani H, Satou N, Satou J. Ciinicai evaluation of two
posterior composite resins retained with bonding agents.
J Prosthet Dent 1989;62:627-632.
570
11. Van Meerbeeit B, Peumans M, Verschucren M, Gladys S,
Braem M, Lambrechts P, Vanherle G. Clinical status of ten
denlin adhesive systems. J Dent Res 1994;73:1690-1702.
12. Prati C, Chersoni S, Ferrieri P, Mongiorgi R, Checchi L
Handling of bonding agents: Clinical procedures. In:
dairOroiogio GD, Prati C (eds). Factors Influencing the
Quality of Composite Restorations-Theory and Practice.
22-23 Nov 1996, Bologna, Italy. Como, Italy: Ariesdue,
1996:55-83.
13. Aitimoto N, Takamizu M, Kohno A. Ciinicai evaluation of
a seif-etching primer system-Seven-year results. Jpn J Conserv
Dent 2000;43:1271-1280.
14. Yoshiyama M, Matsuo T, Pashley DH. Durability of bond
strengths to dog dentin in vivo [abstract 201]. J Dent Res
1998;77:657.
15. Sano H, Yoshikawa T, Pereira PNR, Kanemura N,
Morigami M, Tagami J, Pashiey DH. Long-term durability of
dentin bonds made with a seif-etching primer, in vivo.
JDent Res 1999;78:906-911.
16. Cox CF, Bergenhoitz G, Fitzgerald M, Heys DR, Heys RJ,
Baker JA, et al. Capping of the dentai pulp mechanicaily
exposed to the oral microflora-A 5-week observation of
wound healing in the monkey, j Oral Pathol 1982;11:
327-339.
17 Van Meerbeek B, Willetns G, Celis JP, Roos JR, Braem M,
Lambrechts P, Vanherie G, Assessment by nan o-in dentation
of the hardness and eiasticity of the resin-dentin bonding
area. J Dent Res 1993 ;72:1434-1442.
18. Aliimoto N, Momoi Y, Takamizu M, Kohno A. Evaluation
of mierohardness on resin impregnated iayer and its sur-
rounding area by nano-indentation. Jpn J Conserv Dertt
1996:39:556-562.
19. Saito T, Toyooka H, Matsuda K, Yamauchi M, Crenshaw
MA. In vitro mineral induction by insoluble dentin matrix-
A role of phosphate group and carboxi'l group on mineral
induction. Jpn J Conserv Dent 1997;40:1461-1468.
20. Peachy LD. Eiectron microscopic observation on the accu-
muiation of divaient cations in intramitochondrial granules.
I Cell Biol 1964:20:95-109.
21. Frazier PD, Nyien MU. Biophysicai Studies of Calcium
Transport in Mineralizing Tissues. Presented at the 4th
International Congress of Histochemistry and Cyto-
chemistry, Kyoto, Japan. 21-26 Aug 1972.
22. Weinstock M. Collagen formation-Observations on its
interceiiular packaging and transport. Z Zeilforsch 1972;
129:455-470.
23. Reith EJ. The binding of calcium within the Golgi saceules
of the rat odontoblast. Am J Anat 1976; 147 267-272.
24. Weinstock M, Leblond CP. Synthesis, migration and release
of precursor coliagen by odontobiasts as visuaiized by radio-
autography after =H-proline administration. J Cell Biol
1974:60:92-127.
25. Sayegh FS, Davis R, Solomon GC. Mitochondrlal role in
ceiiuiar mineralization. J Dent Res 1974 ;58:581-587.
26. Granstrom G, Linde A. ATP-depettdent uptake of Ca^* by a
microsomai fraction from rat incisor odontobiasts. Calcif
Tissue int 1981:33:125-128.
27 Beveiander G, Nakahara H, The formation and mineraliza-
tion of dentin. Anat Rec 1966;156:303-324,
28. Tatsumi T. Physiologicai remineralization of artificiaiiy
decaicified monkey dentin under adhesive cotnposite resin
restoration. J Stomatoi Soc Jpn 1989;56:47-74,
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