^ y|Report^

Pulp-dentin biology in restorative dentistry.

Part 2: initiai reactions to preparation of teeth
for restorative procedures
Ivar A. Mjor, BSD, MSD, MD, Dr Odont'

Pulpal compiicaficns involving infiammation, degradation, and necrosis are fhe result of a series of frau-
matic injuries. Tfie resforafive denfisf musf minimize ffie trauma fo denfin and pulp inflicfed during ciinical
procedures, including fhaf infiicfed during fooffi preparation. Part li of tfiis series discusses ffie structurai
and physiologic changes in fhe pulp-denfin compiex fhaf result from crown and oavity preparation and the
ciinicai implication of fhese changes. (Quintessence Int 2001 ;32:537-551¡

Key words: denfinal fluid, hybrid layer puip-denlin physiology, odonfoblasfic displacemenf, smear layer,
tooth preparation

be eftects of restorative procedures on dentin and
T pulp represent a combined response to the prepa-
ration and to the restoration. Long-term effects of
preparation events alone are difficult to assess,
because the preparation will bave to receive a provi-
sional or permanent restorative material or be left
exposed to the oral environment. Beeause no restora-
tive material exists that is truly inert in a biologic
sense, and because preparations left open to the oral
environment will accumulate debris and bacteria, tbe
only way to evaluate structural cbanges in human
dentin and pulp of a cavity or crown preparation is to
extract tbe teetb immediately after tbe procedure is
completed. Nondestructive, physiologic tecbniques
may also be used in experimental studies on animals.
In 1955, a special experimental design was
attempted to evaluate the long-term effect of cavity
preparation.' Cavity preparations with a separate cen-
tral deep cavity were prepared in premolars in chil-
dren. The deep part was covered by a gold plate sealed
in with zinc pbospbate cement. Tbe teeth were
extracted after observation periods ranging from 15
minutes to 4 weeks. Histopatbologic examination of
the pulp showed that tbe reactions subjacent to tbe
deep cavity were the same as in teeth witb cavities
filled witb zinc oxide-eugenoi cement or gutta-percha.
'Professor, Academy 100 Eminenf Scholar, Department of Operative Den-
tistry, LInwersity cf Florida, College of Dentistry, Gainesville, Florida;
NIOM, Scandinaviar Institute of Dental Materials, Haslum, Norway.
Reprint r e q u e s t s : Dr Ivar A. Mjör, University of Florida, College ol
Dentistry. PO Box 100415, Gainesville, Florida 32610. E-mail: imjor©
denial.ufl edu crinijor@nioni.no
This is one of seven articles in a series emptiasizing a biclogic approacti to
restorative dentistry tfirough an understanding of the pjlp-denbri complex.
The eariy reactions included displacement of odonto-
blastic nuclei into the dentinal tubules. After 4 weeks,
reparative dentin had formed subjacent to the cavit\'.
Tbese results are considered to be a combined
effect of two difterent procedures: restoration of a cav-
ity with zinc phosphate cement and preparation of an
open cavity.' Apphcation of current knowledge indi-
cates that the initial observations may have resulted
from tbe higb interstitial fluid pressure in tbe pulp.
Provided tbat the dentinal tubules are patent, cavity or
crown preparations cut into tbe high-pressure area of
the vital pulp can permit dentinal fluid to leak out.
This leakage may be tbe most important effect of cav-
ity and crown preparation, and it will be dealt with in
some detail in this article.
Any pulpal and dentinal cbanges that result from
tbe preparation can affect the evaluation of reactions
to the entire restorative procedure, including possible
toxic and allergic reactions to tbe restorative material
or to bacteria and salivary components. Much atten-
tion has focused on bacteria present on tbe prepared
surface and those at tbe tootb-restoration interface.
What actions to take against tbese bacteria is a con-
troversial topic, but it is generally accepted tbat bacte-
riostatic sealing of restorations is important in restora-
tive dentistry to prevent bacterial leakage. However,
extensive clinical experience bas sbown that contami-
nation of dentin during cavity and crown preparations
has no major effect on tbe outcome of tbe treatment,
and it may stimulate defense mecbanisms in tbe pulp-
dentin organ. Long-term maintenance of a healthy
pulp is a result of atraumatic preparation and the use
of biologically acceptable restorative materials that
can seal the tootb-restoration interface to prevent or
minimize bacterial leakage.

Quintessence International 537

 Teetb tbat are to receive restorations bave or bave
had primary caries lesions, are worn, or bave fractured
because of trauma; many restorations are placed
because of failure of a previous restoration. All tbese
conditions result in changes in the dentin and pulp.
Intact teetb to be used for fixed partial denture abut-
ments may also be involved. Thus, tbe condition of tbe
teetb prior to cavity and crown preparation can be
bighly variable.
In a researcb setting, the evaiuation of pulpal and
dentinal responses to cavity or crown preparation
must be based on tbose responses tbat occur after
preparation of intact teeth from young individtials,
preferahly newly erupted teeth. Tbis selection of teetb
is necessary because the norrnal structure of these
teeth is well estabiisbed, and any deviation in strtic-
ture observed after tbe preparation can tben be attrib-
uted to the specific procedure. Wben a preparation
tecbnique that will not induce cbanges according to
tbe metbod of evaluation employed bas been estab-
lished, tbis tecbnique can be used to prepare cavities
and crowns for subsequent testing of reactions to
restorative procedures, including agents applied to the
dentin, sucb as cleansers, disinfectants, acids, bonding
agents, and restorative materials.
Tbe initial reactions tbat will be described and dis-
cussed in this article will be limited to procedures
immediately preceding tbe restorative pbase of tbe
treatment. Most studies on reactions to tbe cutting of
dental bard tissues bave been carried out witb rotary
instruments using different types of burs, but air abra-
sion and iasers bave also been used. Sucb techniques
bave not received \vide use in dental practice, but they
will be briefly reviewed in this article as alternative
preparation metbods.
Histologie cbanges associated with cavity and
crown preparation can be evaluated witb microscopic
techniques. Pbysiologic changes can be assessed witb
techniques sucb as histocbemical demonstration of
neurogenic components, blood flow measurements, or
recording of interstitial tissue fluid pressure.
FORMATION OFTHE SMEAR LAYER
The normal structure of dentin comprises mineralized
intertubular and peritubtilar dentin and dentinal
tubules (Figs 1 and 2) containing odontobiastic
processes, or their remnants, and tissue fluid, often
referred to as dentinal fluid. If the surface of cut
enamel and dentin is examined after preparation with
band instruments or burs, no structural details sucb as
cut dentinal tttbules {Fig 3) or enamel prisms will be
visible, even at high magnification. All such details are
obscured by a covering layer of cutting debris from the
538
mineralized tissues.^ Tbis grinding debris consists of
ground components of enamel and intertubular and
peritubular matrix, including any content of tbe dend-
nal tubules, mixed with water, dentinal fluid, and often
saliva. Tbis layer is less than 2 jim thick and is termed
tbe smear lay er.^-^
Because tbe dentinal substrate differs as a result of
age-related changes, caries, dentinal sclerosis, and
restorative procedures, tbe smear layer can vary in com-
position.-'^ If the prepared dentinal surface bas open
tubules, small plugs of debris may extend into any open
dentinal tubule. The smear layer reduces tbefluidflow
from tbe dentin and decreases dentinal permeability.^''
Tlie composition of tbe smear layer varies, depend-
ing not only on tbe substrate but also on tbe type of
bur used. If a higb-speed dental engine is used, the
smear layer will be tightly burnished to tbe prepared
surface. Tbe smear layer cannot be completely
removed by a water spray (Fig 4) or by scrubbing, but
it will dissolve during acid-etcbing procedures. Acid
etcbing demineralizes tbe smear layer and tbe per-
itubular and intertubuiar dentin of tbe prepared sur-
face. It leaves the tubules wide open (Fig 5).
The smear layer may also be removed by the appli-
cation of pumice to the prepared surface. Tbis proce-
dure removes tbe smear layer and leaves the smear
plugs in the openings of the tubules in place^ (Figs 6
and 7). A similar selective removal of the smear layer,
leaving the smear plugs intact, can be achieved by the
use of etbylenediamine tetra acetic acid [EDTA) for
cavity cleansing.'
Tbe formation of the smear layer is a pbysical
process and not a biologic reaction per se. However, it
does bave clinical implications that must be dealt with
in a biologic contexL
Tbe smear layer is not a stable structure, and it
must be removed in order to obtain optimal cbemical
and mecbanical bonding between restorative materials
and tooth structures. This demineralization will allow
resin to infiltrate the tubules and tbeir branches, as
well as the collagen mesh of tbe intertubuiar matrix
and the collagen in tbe walls of the tubules exposed by
tbe acid {Fig 8).
Tbe presence of a smear layer can be beneñcial by
physically reducing the flow of fluid through dentin
and thus decreasing its permeability. This reduced
flow of dentinal fluid may have a protective effect on
the puipai tissue. The smear layer may also impede the
entry of bacteria into the cut dentinal tubules."^ An
alternative to removal of tbe smear layer for bonding
to mineralized dentai tissues is to incorporate it as an
integral part of tbe adhesive system. Materials for such
bonding tecbniques bave been mariieted.
Routine restorative procedures that do not include
acid etching are performed witb the smear layer in
Volume 32, Number 7, 2001
 Mjör •

Fig 1 í,---:-^ -.: =:ectron nucrûgrapn oí fractured coronal human
dentm snowing a longitudinal view ol ene dentinai tubuie with dis-
tinct peritubular dentin (PT) lining the tubule. Note the openings in
the wail of the tubuie tor numerous branches Irom the odontoblas-
tic process. (ID) Intertubular dentin (Original magnifioation
X7,400,)
Fig 2 Scanning eiect'O' • i_"_ ;-=!L" I.I -_!;•"•"! reraiizea njman
dentin showing remnants ol the odonloblaslic process (OP) in a
dendnal tubule. The peritubuiar dentin is lost during demineraiiza-
tion. (ID) Intertubular dentin. (Original magnifioation x t .250.)

Fig 3 Sr.íii.íiing eiectron micrograph oí
denîin subjected to high-speed cutting.
Some loose debris is seen, but no
dentinai tubules can be discerned.
(Originai magnifioation X3.000.)
Fig 4 Scanning eiectron micrograph o!
dentin subjeoted io higfn-speed cutting
and subsequentiy washed with a water
spray. No dentinai tubuies can be dis-
cerned, but less debfis is present than
in Fig 3. (Original magniücafion
x3,000.)
Fig 5 Scanning eiectron micrograph ot
ilie ground dentin surface after it has
been treated with 35% phosphoric aoid
for 60 seconds and washed with a water
spray. Note the open dentinai tubules.
The perilubuiar dentin has been dem-
ineraiized. (Original magnitication
x3,000.) (From HOrsted-Bindslev P, fvljör
iA (eds). Modern Concepts in Operative
Dentistry. Copenhagen: Munksgaard.
1988. Reprinted with permission )

FTg 6 (lefl) Scanning electron micro-

graph of ground dentin atter the surface
has been polished with pumice in a rub-
ber cup. Smear plugs (SP) are present in
the openings of the tubules. One dentinai
tubule (DTI does not have a smear plug:
It was probably lost during preparation of
the specimen. (Original magnification
>:3,000.) (From Hörsted-Bindslev P, fwijör
IA (eds). Modern Concepts in Operative
Dentistry. Copenhagen: fvlunksgaard,
1988. Reprinted with permission.)

Fig 7 (right) Scanning electron micro-

graph of undemineralized dentin, sub-
jected to cavity preparation, showing a
longitudinal view of a dentinai tubule (DT)
with a smear plug (SP). (Courtesy of Dr
M. Ferrari: original magnification x7.5iX).)

539
Quintessence International

Fig 8 Scanning electron micrograph of prepared and acid-
elched dentin Note the inlerwoven libers on the cavity (CAl flooi
and in the wall of the denlinal tubule (DTl (Courtesy ol Dr M
Ferran: original magnification x7,5QD.l
place. Because a sterile technique is rtot used, it is
likely that bacteria can be found in the smear layer,
even if a rubber datn is ernployed. This situation has
raised questions about the need to sterilize cavity
preparations prior to restoration." '- Any antibacterial
treatment applied to the prepared surface may alter
the conditions for adhesion and may lead to pulpal
reactions. Many liners, bases, and luting cements have
antibacterial properties; it is also likely that the acid-
etching procedure used in conjunction with adhesive
restorative techniques exerts an antibacterial effect.
These various agents are likely to be supplemented by
antibacterial defense mechanisms in the pulp, because
it has been claimed that vital dentin resists infection.'^^"^
REACTIONS TO
CAVITY AND CROWN PREPARATION
Structurai changes
It has long been established that preparation tech-
niques are available that cause nu or few histologie
changes in teeth after evaluation of demineraiized sec-
tions stained with hematoxylin and eosin''-'' (Fig 9).
Adequate cooling of a bur cutting at high speed is
essential to prevent histologie changes in the dentin
and injury to the underlying odontoblastie region of
the pulp- Temperature increases can cause severe
injury to the pulp, and coolants should always be used
540
during cavity and crown preparation. Care must be
exercised to ensure that the water spray effectively
coois the bur at the cutting surface. "Shadowing"
effects by the tooth may prevent the water spray from
reaching the hur (Fig 10). It is difficult to avoid histo-
logie ehanges to the underlying pulp if a crown prepa-
ration is performed at high speed, even if an adequate
water cooiing system is employed.' Intermittent cutting
using light handpiece pressure can minimize tempera-
ture increases during cavity and crown preparation.
If histologie sections showing so-called harmless
cavity preparations are scrutinized, a separation of the
dentin and pulp is often found, localized to the tubules
exposed by the eavity preparation (Figs 11 and 12).
This separation is likely to be a histologie artifact, but
because it is often limited to the tubules exposed by
the cavity preparation, it is probable that some injuri-
ous changes that predispose to the separation have
occurred in the dentin or in the pulp-predentin region.
The injury inflicted on dentin and pulp when cooling
of the bur is inadequate during cavity and crown prepa-
ration of dentin can lead to displacement of odontoblas-
tic nuclei into dentinal tubules {Figs 13 and 14). Similar
histologie changes can occur if the dentin is dried exces-
sively after the preparation is eompleted. Marked disor-
ganization in the organelles of the odontoblasts and in
tbe adjaeent cells can also be observed (Fig 15). These
responses must be regarded as gross reactions to injury.
Overheating or burning of the dentin during erown and
cavity preparation are tbe most common reasons for
displacement of cells into the dentinal tubules and for
disruption of the eontents of the tubules.
Burning of the dentin was a frequent oecurrence in
the early days of restorative dentistry. Ahhough low
speed was used, considerable pressure on the bur pro-
duced frictional heat. Present-day high-speed equip-
ment is designed to supply adequate cooling. However,
care must be exercised to ensure that the water jet
actually reaches the cutting edge of the bur at all times
and that no part of the preparation prevents the water
jet from reaching the cutting part of the bur,'^ as seen
in Fig 10. If dentin is overheated or burned during
cavity or crown preparation, a color change will be
visible in the margin of the preparation, as seen on his-
tologie sections when certain stains are used'^ (Fig 16).
Less dramatic changes than displacement of odon-
toblastic nuclei can be demonstrated by staining sec-
tions of prepared teeth to show the presence of glyco-
saminoglycans. These dyes stain componetits that are
located intratubularly in a specific segment of the
dentinal tubule in newly erupted teeth, and they may,
therefore, be used as a marker for changes occurring
in the content of the tubules (Fig 17). The application
of these special stains to demineraiized sections of
newly erupted teeth can reveal distinct histologie
Volume 32, Number 7, 3001

Rg 9 Photomicro g rap h of a demineralized section ot a newly
empted premolar that was extraoted immediately atter cavity (CA)
preparation with a bur supplied with an effective water cooling
system, (tíotteü line) Extent of the tubules opened by tfie cavity
preparation. Note the intact odontoblastio (O] layer and distinct
cell-free zone adjacent to it. (Hernatoxyhn-eosin stain; original
magnification x220.)
Fig 11 Photomicri,g[apr, or a C5-ity (CA) prepared prior to
e>araction oi the tooth, (dotted tine) Extent of the tubules cpehed
by the oavity preparation. An intact odontoblaslic (O) layer is
[weseht subjacent to the cavity, but the cell-free zone is not dis-
tinct. The separation (S] between the predentin and the odonto-
blastic layer is probably a histoiogic artifact: however, because it
is limited to the tubules opened by the cavity preparation, it does
signify that some changes had resulted from the preparation.
(Hematoxylin-eosin stain: original magnification x90.)
Quintessence International
Mjör •
Fig 10 Head ol a contra-angled handpiece with a bur in place
and with the water spray turned on. The water jet does not reach
the working part of the bur because the tooth intervenes. (Cour-
tesy of Dr K. Langeland.)
Fig 12 Higher magnification of the separation (S) between the
predentin (PD¡ and the cdontoblastic (O] layer as a result ol cavity
preparation similar to Ihat shown in Fig 11. (dotted iine) Extent of
the dentinal tubules (DT] opened by the cavity preparation.
(Hematoxylin-eosin stain; demineralized section, original magnifi-
cation x350.]
541
• Mjor

Fig 13 Pholomicrograph of a üemineral-
ized ^ec'.ion Slowing odontobiastic nuclei
{ON] displaced into the dentin as a result of
cavity préparation with ¡nadequafe cooling
of the bur. The odontoblasfic layer is dis-
rupted and vacuoiized. (Hematoxylin-eosin
stain; original magnification x60.]
Fig 14 Pfiotomicrograpfi of a demineral-
ized section showing odontoblastic nuciei
(ON) dispiaced into tubuies of the pre-
dentin (PD) subjacent to a cavity prepara-
tion (Hematoxylin-eosin stain: original
magnification x90D.)
Fig 15 Electron micrograph of jncJ
aiized dentin siiowing damaged and disoi.
ganized cytoplasmic components from
odonloblasfs displaced into the prederlin
(PD) foliowing (he grinding of a tat molar.
(ívl¡ mitochondrium; (REB) rough endoplas-
matio reticulum. (Courtesy of Dr O.B.
Sueen; original magnification x15,000.)

cbanges in dentin in which cavities bave been pre-
pared with a high-speed dental engine supplied with
abundant water spray. This altered staining of the
dentin occurs without displacement of odontoblastic
nuclei into the tubules.
The teeth frequently used in such investigations are
intact premoiars that are to be extracted from cbildren
aged 10 to 14 years for orthodontic reasons. The spe-
cific change noted in these sections is based on tbe
presence of intratubular reactive components tbat
have shifted position following preparation of the
dentin. The position of these reactive components
after preparation indicates outward movement or dis-
placement of the tubular contents (Figs 17 to 19), Such
movements occur even if a so-called nontraumatic
preparation technique is used.
The outward movement of the contents of the
tubules is probably a result of tbe exposure of the
dentin for the first time in an otherwise unaffected
tootb. Tbe preparation opens up into a high-pressure
area because tbe normal interstitial tissue fluid pres-
sure of tbe pulp is in tbe range of 5 to 20 mm Hg.'^'^*"
It appears that this displacement of the contents of the
tubules cannot be prevented wben preparations are
made in newly erupted, intact teeth,^' The movement
of the contents of the tubules is dependent on tbe
tubules' being open. No studies similar to those
referred to on newly erupted teeth have been per-
formed on intact teeth of adults or older individuals,
in whom the tubules may be partly or completeiy
obturated by grow'th of tbe peritubular dentin.
Cavity and crown preparations reaching dentin in
newly erupted, intact teeth will expose tubules that are
normally open. Protrusion of the contents beyond the
tubules is sometimes observed in histoiogic sections
(Figs 18 and 19). The reason that protrusion is not
observed from all or most of the tubules may be that
the reactive part of the contents has been completely
extruded through tbe dentin. Furtbermore, a smeai
layer may obturate the opening of tbe tubules anc
block or reduce extrusion of the contents (see Figs 2
and 4). The fact that the contents occasionally pro
trude beyond tbe cut tubules indicates tbat an activi
force has been applied, because capillary forces alom
would fill, but not overfill, the tubules. Tbese change;
cannot be prevented, and they occur even where ni
displacement of odontoblastic nuclei takes place (Fig:
20 and 21). Exudation of fluid from dentinal tubule
after cavity preparation has also been shown througl
replica (impression) techniqttes.^^
The clinical signiflcance of outward movement c
the tubular contents bas not been established. It ha
been suggested that the localized, reactive, and stain
able contents of the tubules a short distance into th
dentin from tbe dentin-predentin border are associate
witb secondary formation of peritubular dentin i

542 Volume 32, Number?, 20C

Fig 16 (left) Demjneralized section show-
ing altered staining (red) ol ttie cavity (CA)
margin ttiat resulted wtien inadequate oool-
ing ot ttie bur led to burning ol the dentin.
(Original magnitication x25.)

Fig 17 (right) Photomicrograph ot a de-

mineralized section ol an occlusal cavity
(CA) preparation on a newly erupted, intact
premolar that was extracted immediately
after preparation. The normal staining pat-
tern ot unattected dentin is shown on the
lett. Note \Ue altered staining ot the dentin
subjacent to the oavity. (Z) Zone wiiti
intratubular reaorive components in unaf-
fected dentin. (Méthylène blue stain, origi-
nal magnification x35.)

Fig 18 (left) Photomicrograph of a de-

mineralized section showing tubular con-
tení protruding into rhe cavity (CA] prepa-
ration. The tooth was extracted immediately
atter the cavity preparation. (Alcian blue
and periodic acid-Schitt stain; original
magnification x900.]

Fig 19 (right) Dentin at the cauity (CA)

margin sfiowing dentinal tubules (DT) with
intratubular staining, which is normally
¡ound only in more puipally located dentin
(corresponding to Z in Figs 17 and 21 i
Note the protrusion ot tubular contents intc
the cavity preparation. (Original magnitica-
tion x900.)

newly erupted teeth." Preparation and restoration of
teeth may, tberefore, have an effect on tbe secondary
development and growth of peritubular dentin.
Tbe reactive components within tbe dentinal
tubules include cytoplasmic constituents, Tbese con-
stituents and any components present within tbe peri-
odontoblastic space, including tissue fluid, apparently
become displaced. The disturbance and redistribution
of tbese cellular constituents (Fig 15) will result in
degeneration of the odontobiastic processes'"-* (Figs
22 and 23). If tbe preparation is restored, no reestab-
lisbment of normal staining reactions is observed-' (Fig
24). However, reestablishment of the normal staining
pattern has been shown to occur if coronal dentin is
exposed to the orai environment in shallow, self-
cleansing facets for at least 7 days^' {Fig 25).
The ciinical signiflcance of any change following the
displacement of odontobiastic nuclei and/or the con-
tents of the tubules has not been fully estabiisbed, but
it is likely to have an effect on tbe physiology of the

543
Quintessence International
 Mjör
Fig 22 Electron micrograph ot an undemineraiized section
shewing a dentinai tubuie (DT) with the remains ot a disintegrated
odontobiastic process. (Ve) Vesicles coated with dark granuies.
(Courtesy of Dr O.B. Sveen: originai magnification x 13,700.)
544
Fig 20 (lell) Photomicrograph ot a de-
mineraiized section ot a cavity (CA) prepa-
ration, prepared with an abundant water
ccDiing of the bur The odontobiastic (0)
iayer subjacent to tne cavity remained
intact and no dispiacement of odontobiast
nuclei inio the dentinai tubules has
occurred (dotted iine) Extent of the cavify
preparation (Hemato>7lin-eosin stain: origi-
nai magnification x220.)
Fig 21 (right) Photomicrograph ct (he
seclion adjacent to the one shown m Fig
20, Note the difference in staining ot the
denlin subjacent to the cavity (CA) prepa-
ration and that ol unaffected dentin to the
left of Ihe dotted line, which delimits (tie
extent of the cavity preparation. (Z) Zone
with intratubular, stainabie components as
part of the normal staining pattern of
dentin. (Toiuidine blue stain; originai magni-
lication X220.)
Fig 23 Eiectron micrograph ot an undemineralized section
showing a dentinai tubule (DT) with ceiluiar fragments undergoing
necrosis. The iarge, light, circular areas within the tubuies may be
iipid dropiets. (Courtesy of Dr O.B. Sveen: originai magnification
X11,200.)
Voiume 32, Number 7, 2001

Fig 24 P^otomlc^og^apn of a demineraiized section showing
^tered staining of dentm subiaoent to oavities ttial nad been
restored *ith calcium hydroxide and amalgam ¡CA-I] and zinc
oxide-eugenol oement (CA-2) 4 days prior to extraction of me
tooth. The zone (Z] with stainable inlratubular components is
missing sub|acent to the oavities. (dotted iines) Extent of the
tubules opened by the cauity preparation. (Alcian blue stain: angi-
ng magnification xd5 ¡
affected dentin. The intratubular changes may be the
start of a "dead tract" reaction.-* The formation of a
dead tract may be dependent on the disturbance of the
contents of the tubules and subsequent formation of
tertiary dentin subjacent to the affected dentinal
tubules. However, a certain degree of trauma to tbe
odontoblasts and their processes must take place
before a dead tract will develop, including the forma-
tion of an atubular "hyaline zone" between the physio-
logic secondary dentin and the tertiary dentin. This
hyaline zone corresponds to the interfacial dentin.^'
and the tertiary dentin compares to reparative dentin.^*
Physioiogic changes
Immediate vascular responses have been demon-
strated to result from the grinding of dentin. Olgart-^^"
summarized the findings from a series of experiments
on cats over a 25-year period that included common
clinical procedures using neurophysiologic and hemo-
dynamic techniques. These procedures comprised
grinding of dentin and percussion of the teeth. Brief
grinding (1 second, 3 times) of feline canines with a
diamond bur flushed with saline at 6,000 rpm caused
Quintessence Internationaf
Mjör •
Fig 25 -'hoiümicrograph of a demineraiized section. The coronal
deniin had been exposed to the orai environment loilowing the
grinding ol a facet (FA) on the newly erupted premolar 35 days
before the tooth was extracted. The normai staining ot denlin in
the zone (Z] with siainable intratubular components is found both
subjacent to the facet and in the unaffected dentin (Toluidine blue
slain; originai magnification x35.)
an instantaneous increase in biood flow (Fig 26). The
blood flow in denervated contralateral teeth exhibited
significantly smaller and much delayed responses.
Grinding halfway into dentin caused a 530'0 increase
in blood flow lasting for about 10 minutes. Further
grinding into deeper layers of dentin caused only
minor differences in the magnitude of the response.
The effect of anesthesia on the blood flow was illus-
trated in a series of experiments using ultrasonic stitit-
ulation-^ (Fig 21). The findings from these experiments
were interpreted to support the concept that extensive
branching of pulpal nerves is associated with func-
tional connections to periodontal tissues.
In another experimental series, using teeth with
intact enamel, a setup imitating the percussion of teeth
instantly produced a 30% increase in blood flow last-
ing about 2 minutes. The increase in blood flow fol-
lowing percussion was smaller tban tbat after tbe
exposure of dentin by grinding, perhaps because of the
lack of exudation of dentinal fluid from the intact
teetb. Tbese investigations were not supplemented by
bistologic studies, which would have allowed correla-
tion between the blood flow recordings and the degree
of movement of the contents of the tubules, including
545

Fig 26 Pulpal blood How, expressed as a percentage of laser
Doppler flowmetry (LDF) following grinding (3 times, 1 seccnd
each] with a round diamond bur at 6,000 rpm. Grinding in the
Inner hall of the dentin (btack diamond) causes a rapid increase
in pulpal blcod flew in a normal, innervated feline canine (upper
curve) In the denervated contralateral canine, the corresponding
response is delayed and smaller (lower curve). (From Olgart^^ and
the Finnish Dental Society Appolonia. Reprinted with permission.)
displaccmcnf of odonfoblasfic nuclei info the tubules.
Such displaccmcnf is more likely to occur following
exposure of dentin than in teeth with intact enameL
because fluid flows through enamel much less than
through exposed dentin.''"
Crown preparations made with a high-speed bur
without water spray have been sbown to decrease
blood flow in the pulp of dog canines.^' The magnitude
of the decrease in blood flow was dependent on the
remaining dentin thickness. If the preparation reached
the inner third of dentin, whicb was estimated to ieave
about 1 mm of remaining dentin, the blood flow was
reduced by 90^/0 añer 1 hour. Preparation to tbe same
depth bad negligible effect on the pulpal blood flow
wben abundant water spray was used to cool the bur.
Dry preparation balfway into dentin resulted in a sig-
niflcant increase in blood flow through shunt vessels,
especially those in the apical part of the teeth.
Impressions of the prepared surfaces of rat teeth,
which involved tbe use of a copper band witb warm
wax, caused severe fluctuations in blood flow; minimal
changes were noticed if rubber-based impression
materials were used. A further reduction in blood flow
resulted from epinephrine in anesthetic solution. Thus,
both the combination of dry preparation with high
speed and the use of anesthetics with a vasoconstric-
tor are considered to be particularly hartnful to the
546
SO T Ultrasonic stimula on
er:) Before local ar esthetic
1^ ^ Atter local ane sthetic
•3 60
œ
£ 40
1 1
3 20
8 •
Low
6
High
1 11
High
Fig 27 The effect ot injection of local anesthetic solution on the
blood flow following ultrasonic stimulation of a feline canine.
Periapical injection with a local anesthetic solution, leaving the
vasodilator response to electrical tooth stimulation intact (A), abcl-
ished the vasodilator response to low-amplitude ultrasonic stimu-
lation but not that induced by high-arnplitude stimulation.
Additional subperiosteal apical injections blocked the intrapulpal
nerves (B), and this procédure also reduced the response to high-
amplitude ultrasonic stimulation. Numbers in bars indicate tiie
number of experiments performed. (LDF) Laser Doppler llow-
metry. • = P < .0005. (From Olgart^ä a^d the Finnish Dental
Society Appolonia. Repnnted with permission.)
pulp. These investigations were not supplemented by
histoiogic studies. Thus, no correlation with struijtural
changes can be made. However, bistologic cbanges in
tbe pulp and dentin following complete crown prepa-
ration have been sbown to occur, and they are consid-
ered difficult to avoid.*
The vasoconstriction noted after cavities are pre-
pared deep in dentin without a coolant may be due to
inhibition of sytnpatbetic nerve stimulation.^'''^'"' It has
also been sbown tbat stimulation of cervical sympa-
tbetic nerves results in a significant decrease in fluid
flow through dentin. Such changes in fluid flow may
increase the rate of diffusion of agents from the denti-
nal surface to the pulp.^-^ Conversely, an increased
flow from the tubules, eg, because of inflammation in
the pulp, may prevent or reduce the diffusion of bacte-
ria and toxic agents into the pulp.'^
It is evident that the dentinal fluid plays a central
role in dentin physiology, ineluding those changes that
result frotn restorative procedures. The peripheral flow
of this fluid following cavity preparation allows
plasma proteins to enter the tubules. Clotting of these
proteins will reduce the functional diameter of the
tubuies and reduce tbe permeability of the dentin."'*
It was believed tbat fibrinogen caused the obstruction
of the tubules, but the presence of fibrinogen has been
difficuh to demonstrate.
Volume 32, Number 7, 20D1
 Tbe presence of serum albumin in the tubules has
been established.^^-"* Cavity preparation into the inner
third of the dentin in intact premolars from children
causes an albumin flux. This exudation of albumin was
markedly reduced after 2 days of exposure to the oral
environment and after another 12 days following the
placement of a zinc oxide-eugenol cement to seal the
cavity.-" The exposed dentin was covered by a tbin coat
of Teflon to prevent the cement from blocking the
tubules in these experiments. Histologie studies of the
pulp showed good preservation of the pulpal tissue
without cellular infiltration. Following bactériologie
challenge, marked cellular infiltration was found subja-
cent to the tubules exposed by the ca\'ity preparation,
including positive immunochemical reactivity for
macrophages. Although the experimental procedures
did not render the dentin impermeable, the reduction in
perméabilité' may be clinically important in preventing
bacteria and toxic agents from diffusing into the pulp.
It is apparent that preparation, impression, and per-
cussion of teeth may result in significant vascular
changes in the dental pulp. These changes are tran-
sient and usually resolve without clinical complica-
tions. Whenever the blood flow is impeded in the
pulp, locally or systemically, the reaction may be the
start of an adverse process. If tbe vascularity increases,
tbe reaction may be looked on as a defense mecha-
nism to initiate preparedness for subsequent insults.
The odontoblastic layer comprises closely packed
cells that often appear pseudostratified in coronal
dentin. A number of junctional complexes link the
odontoblasts together. This layer of cells exhibits sev-
eral characteristics similar to those of an epithelium.^^
ït may act as a barrier and provide a protective efl'ect
by preventing macromolecules from passing from the
pulp into the dentin. Tbis barrier is often disrupted
during crown and cavity preparation and tbe physio-
logic reactivity in the region will change. It is likely
that the junctional complexes become reestablished,
but the nature and significance of this repair process
are unclear.
Mechanisms governing displacement
of odontoblasts and tubular contents
Displacement of odontoblastic nuclei into the dentinai
tubules is a phenomenon that has long been recog-
nized. It occurs regularly subjacent to marks on roots
from the beaks of extraction forceps-" but may also
occur for a variety of other reasons, including cavity
and crown preparation. Considerable attention was
paid to tbis pbenomenon when high-speed dental
engines were introduced in the late 1950s. The phe-
nomenon has also been described as "aspiration" of
odontoblastic nuclei into dentinal tubules,"'^' but tbe
Quintessence International
term displacement of odontoblasts'^ has come into
common use, because it is not suggestive of any spe-
cific mechanism for its occurrence. Other celis present
subjacent to tbe tubules exposed by cavity preparation
can also be displaeed into tbe tubuies under extreme
conditions.'^
Provided that an adequate water spray is used dur-
ing tbe cavity preparation, displacement of nuclei wifl
not occur {see Figs 9 and 20). Intermittent, low-speed
cutting of dentin witb light pressure and without water
spray is routinely performed, especially during
removal of the final carious dentin in deep cavities. If
performed with care, this "excavation" with a large
round bur used witbout water spray is considered
acceptable treatment, but cavity depth is an important
modifying factor.-*"-** Reduction in remaining dentin
thickness makes the pulp more vulnerable to injury
from cavity and crown preparation trauma.
Electron microscopic studies of the pulp-predentin
interface after cutting of dentin^^ bave revealed
displaced cellular contents and odontoblastic nuclei
in some of tbe tubules. A number cf morpbologic
changes bave also been noted, including intraceliular
disorganization and rupture of tbe nuclear membrane
(see Figs 15, 22, and 23). Tbis process takes place
quickly and causes disruption of the odontoblastic
layer. Lysis of the cellular elements takes place over
time, and an inflammatory reaction occurs in the adja-
cent pulpal tissue. After about 20 days in human teeth,
the displaced cells will have disintegrated, and nuclei
cannot be discerned in the dentin.'=
Tbe mechanisms involved in displacement of odon-
toblastic cell bodies into tbe tubules and in displace-
ment of the contents of the tubules are not fully under-
stood. The displacement of nuclei is considered to be a
more exireme reaction than that limited to movement
of tubular contents. Many theories have been put for-
ward to explain the displacement of nuclei into the
tubules and the disorganization of the contents within
the dentinal tubules. Because displacement of odonto-
blastic nuclei is regularly found corresponding to the
forceps marks on the root following tooth extraction,
mechanical distoriion of tbe dentin is a likely explana-
tion for the pbenomenon in that context.
Tbe distortion of teeth during extraction may be
transmitted through the body of the pulp. If extraction
forces are transmitted via the pulp, fluid flow is more
likely through exposed tubules than through tubules
covered by enamel and cementum. No such distortion
occurs when animals are killed by an overdose of
anesthesia and tissue blocks witb the teeth in place are
dissected after histoiogic fixation, but displacement of
odontoblasts can sfiU occur.^-' Furthermore, a compa-
rable distortion is unlikely to occur during the prepa-
ration of teeth and foliowing excessive drying of the
547
 prepared dentin. Thus, tnechanical distortion of pulpal
tissue alone cannot explain the phenomenon, but it
may be a contributing factor.
Other theories for displacement of tubular cotiients
include evaporation of fluid from the prepared surface,
especially from heat generated during the preparation,
marked differences in osmotic gradients,''^'*' and
chemotaxis from toxic agents on the dentinal surface.
Evaporation of the contents of tubules occurs follow-
ing preparation without adequate cooling of the bur or
as a result of excessive drying of the prepared surfaces.
Capillary forces will then replace lost dentinai fluid
with the interstitial fluid in the pulp. A buildup of
intrapulpal pressure due to inflammation has also been
suggested,-" but bistopatbologic evidence of inflamma-
tion is not an immediate response to crown or cavity
preparation. On the other hand, the increase in blood
flow as an immediate reaction to the grinding of
dentin-' is likely to increase the tissue fluid pressure
locally.
More than one mechanism may be in operation to
explain displacement of tbe contents of dentinai
tubules, depending on the clinical situation. It is likely
that the normal high interstitial fluid pressure in tbe
puip plays a role in displacing the tubular contents, at
least in teetb wbere tbe dentin is exposed and the
tubules are open and not obturated by mineralized
deposits. Because extraction forceps denude the root
dentin, the pressure gradient may also be a factor in
this connection.
Irrespective of the mechanism invoived, it is diffi-
cult to explain part of the movement of tubuiar con-
tents induced by cavity preparation in vitro, both prior
to and after fixation of the teeth for histologie prepara-
tion.""^'^^ These findings suggest that physical and
possibly chemical forces play a major role in tbe dis-
placement of tbe contents of the dentinal tubules.
The outcome of displacement of cytoplasm, nuclei,
and otber cellular components is a disintegration and
degeneration oí the contents of the tubules^"* (see Figs
22 and 23). Waste products will cause some degree of
inflammatory reaction in tbe pulp; based on bistologic
and clinicai experience, the inflammatory response will
usually be followed by heaiing. However, this type of
additional trauma is unnecessary and should be pre-
vented. In some teeth, cavity or crovm preparation may
be tbe additional trauma tbat results in pulpal compli-
cations in an already compromised pulp or hypersensi-
tivity and discomfort for the patient after treatment.
A number of factors affect the pulp-dentin repair
processes following restorative procedures, including
remaining dentinal thickness, age of the patient, factors
related to cavity dimensions, and possihly tbe release of
growtb factors.''" Tbese issues will be discussed in detaii
in a later article on reactions to restorative procedures.
548
ALTERNATIVE PREPARATION METHODS
Rotary instruments witb stainless steel, tungsten car-
bide, and diamond burs of different shapes and sizes
are routinely employed to prepare cavities and
crowns. Alternative cutting methods include air abra-
sion and lasers.
Air abrasion equipment tbat uses abrasive dust has
been developed for cutting tooth structure, but for sev-
eral reasons tbe clinical application of tbis technique
never became popular. It did not allow tactile sense
during tbe cutting procedure, and the abrasive dust
obscured the field of operation. The dust may also be
inhaled and therefore represents a potential health
problem for tbe patient and tbe operator.
During tbe last few years, air abrasion equipment
has been promoted for cleaning pits and fissures prior
to tbe application of sealants. The need for such clean-
ing has not been demonstrated. On the other hand,
cleaning of pits and fissures can aid in the diagnosis
of occtusal caries. However, the inherent problem with
dust management remains; because tbe cleaning proce-
dure only calls for removai of debris, the equipment
should be used intermittenfly and for short periods. The
iacii of tactile sense, therefore, is of minor importance.
Laser equipment is based on tbe use of beams of
high light intensity, laser being an acrotiym for light
amplification by stimulated emission of radiation.
Ligbt photons of characteristic wavelengths are pro-
duced, amplifled, and flltered to make tbe laser beam.
Carbon dioxide and neodymium:ytterium-aluminium-
garnet lasers are most commonly used.
The main problem witb laser cutting of hard dental
tissues is tbe generation of beat. Increases in pulpal
temperature of more tban about 5°C may lead to dam-
age. However, laser equipment is not used for cavily
preparation but has a number of potential applications
in dental practice, including coalescence of pits and
fissures to eliminate retention sites for bacteria, desen-
sitization of exposed root surfaces, rougbening of bard
tissue suriaces to promote bonding as an alternative or
supplement to acid etching, vaporization of carious
tissue, and endodontically for vaporization of organic
tissue in the root canal." Limited research on pulpal
reactions to laser cutting of dentin calls for caution in
the use of this technology in restorative dentistry. Tbe
ability of such tecbniques to coalesce deep, narrow fis-
sures is also questionable.^*
FORMATION OFTHE HYBRID LAYER
Acid etcbing of enamel and dentin exposed by prep-
aration, referred to as "tbe total-etch technique,"^^ hgg
become routine treatment in conjunction with adhesive
Volume32, Number 7. 2001

techniques. Acid etching deminerahzes hard tissues
and exposes the organic matrix. The scanty organic
matrix of enameJ is lost during the demineraJization
and subsequent washing. The components of dentin are
selectively demineralized. The most significant expo-
sure of collagen occurs after acid treatment of the inter-
tuhular matrix of the dentin (see Fig 8).
The highly mineralized perituhular dentin deminer-
ahzes quicker than does the intertubnlar matrix. This
demineralization widens the tubules, making them
funnel shaped toward the surface. It exposes collagen
on the wall of the tubules and also uncovers tbe open-
ings of a large number of lateral brancbes tbat may be
important for penetration of resin to acbieve optimal
bonding to dentin.^*
The quality of the demineralized dentin is impor-
tant for adhesion of resin, and the demineralization
should not denature the collagen.=' Phosphoric acid
and citric acid are the most commonly used acid
etchants. The addition of 3% ferric chloride to 10%
citric acid markedly enhances the adhesion to dentin
by preventing the denaturation of collagen. The colla-
gen exposed by acid etching forms an interwoven
mesh of fibers that the resin will infiltrate {see Fig 8). injuries. It is, therefore, the responsibility of the
This collagen mesh infiltrated by resin is referred to as
the hybrid layer.^*^ It is about 5 to 10 ym thick. After and pulp inflicted during all clinical procedures,
polymerization, the resin-impregnated collagen,
together with the resin in the dentinai tubules and
their branches, constitutes tbe adhesion between tbe
dentin and the resin.
The formation of the hybrid layer is basically a
chemical process involving dissolution of primarily
mineral salts and noncollagenous matrix components
followed by diffusion of resin into tbe remaining colla-
gen matrix. Because it bas clinical implications, it is
important that such treatment be considered within a
biologic context. The chemical treatment of dentin
may also release growth factors that may be important
for subsequent reparative processes.'^
Mucb attention has been focused on the degree of
wetness of the hybrid layer at the time of application
of the resin.*''-*^ ¡f ,he hybrid layer becomes too dry,
the collagen mesb will collapse and penetration of
resin will be impaired. To obtain optimal bonding
between the resin and the hybrid layer, the surface
must have an adequate moisture content to prevent
collapse of the collagen mesh. The ideal degree of wet-
ness may vary from one resin-based product to
another. The wetness will certainly difter on the vari-
ous parts of the prepared surface because of the differ-
ent densities and the structure of dentinai tubules in
different locations on this surface." Thus, the instruc-
tions for use of resin-based materials must take these
issues into consideration, and they must be followed
closely to obtain the best possible clinical result.
Quintessence International
Mjöf
Furtber details related to tbe bybrid layer will be out-
hned in a discussion of adbesive restorative techniques
later in this series of articles.
CONCLUDING REMARKS
Structural and pbysiologic cbanges resulting from
crown and cavity preparation in vivo bave been out-
lined in many experimental studies over the last 50
years. A number of biologic reactions bave been
shown to occur. Some reactions are of a physical or
cbemical nature, but tbey also bave biologic and clini-
cal implications. Altbougb some of tbe reactions are
clearly understood, for others the clinical implications
are largely unknown, eg, the displacement of cell
nuclei into dentinai tubules and tbe disruption of tubu-
lar contents following crown and cavity preparation.
Restorative dentistry is possible even if these reactions
are disregarded; however, if restorative dentistry is to
evolve as a biologic science, tbey must receive atten-
tion, clinically as well as in continued research efforts.
Pulpal complications involving inflammation, degrada-
tion, and necrosis are the result of a series of traumatic
restorative dentist to minimize the trauma to dentin
including that occurring during the preparation phase.
ACKNOWLEDGMENTS
A Guest Research Fellowship from the Research Council of Nor^'ay.
parlly in support of the author's Facuky Developmental Leave at
NIOM. Scandinavian Institute of Denial Materials, is gratefully
acknowledged.
The author would also like to thank Dr A. I. Smith, Professor and
Chainnan, and Dr P. E. Murray, Unit of Oral Biology. School of
Dentistry, University of Birmingham. Birmingham, England, for
reviewing Ihe manuscript.
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56. Gwinnett A¡, Tay FR, Pang KM, Wei SHY. Quantitative tners and dental hard tissues—gives clinicians a
contribution of the collagen network in dentin hybridiza- versattle new materiai, useful in a wide array of
tion. Am I Dent 1996:9:140-144. advanced dental treatments. As the first in-depth
57. Mizunuma T. Relationship between bond strength of resin exploration of the sub]ect, this book covers the
to dentin and structural change of dentin collagen during development, present understanding, and future
etching. Influence of ferric chloride to structure of the colla-
research areas of thts multifunctional dental mate-
gen. ] Jpn Dent Mater 1986:5:54-64
rial. A thorough review of the current literature
58. Nakabayashi N. Resin reinforced dentine due to inflltration
rounds otjt the text.
of monomers into dentine at the adhesive interface. [ Jpn
Dettt Mater 1982;1:7S-81. Valuable for students, researchers, atid clini-
59. Zhao S. Sloan AJ. Murray PE, Lumley P), Smith AJ. Ultra- cians seeking a greater understanding of resin
structural localization of TGF-h exposure in dentine hy hybridization of tooth struaure.
chemical treatment. Histochem J 2000;32:489-494.
50. Gwinnett AJ. Moist vs. dry dentin: Its effect on shear bond
strength. Am J Dent 1992:5:127-129.
61. Kanca J III. Resin bonding to wet suhstrate. 1. Bonding to CONTENTS
dentin. Quintessence Int 1992;23:39-41. ! Evolution of Dentin-Resin Bonding
52. Jacobsen T, Soderholm KJ. Some effects of water on dentin 2 Properries of Derttin
bortding. Dent Mater 1995;11:132-136. 3 Acid Conditioning and Hybridization of
63. Mjör IA, Nordahl I. The density and branching of dentinal Substrates
tubules in human teetb. Arch Oral Biol 1996 ;41:401-412. 4 Characterization of rhe Hybrid Layer
5 The Quality of the Hybridized Dentin
6 Ciinical Applications of Hybrid Layer
Formation

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