Review

Recent Advances in the Theory and Mechanism of Adhesive

Resin Bonding to Dentin: A Critical Review
T. K. Vaidyanathan, Jayalakshmi Vaidyanathan
Department of Restorative Dentistry, NJ Dental School, University of Medicine and Dentistry of New Jersey,
Newark, New Jersey 07103

Received 28 March 2008; revised 1 July 2008; accepted 14 July 2008

Published online 29 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31253

Abstract: Dentin bonding issues involving adhesive resins have attracted considerable
research interest in recent years. An important advance due to the ongoing research is the
concept of hybridization of the tissue with primer/adhesive systems. Hybridization involves
permeation of primer monomer into the tissue substrate. Although the mechanism of adhesive
permeation and interaction with tissue may be complex, significant advances have been made.
In systems where etching precedes priming and bonding steps, the Hoy’s solubility parameter
compatibility of the primer formulation with that of demineralized dentin matrix may
determine adhesive permeability. Monomer permeation brings the primer atoms in closer
contact with the substrate atoms, leading to adhesive interactions through van der Waals,
hydrogen bonding, and electrostatic interactions. In self-etch primer systems, stronger
electrostatic interaction between primer monomers and hydroxyapatite has been used to
explain the adhesion process. These interactions have been computer-modeled and analyzed.
Such interactions and subsequent polymerization of the monomer promote improved bond
strength and efficient margin sealing. Incomplete permeation of monomer into the full depth
of demineralized region may, however, leave exposed collagen fibrils and cause nanoleakage of
water into these regions through a 20–100 nm sized marginal gap, leading to subsequent
hydrolytic degradation of these collagen fibrils and the hybrid layer. Microleakage is also a
problem in some single step formulations. In this review, we analyze these current theoretical
and mechanism-related issues of interest in adhesive resin bonding to dentin, and outline the
continuing problems that need to be overcome in the future. ' 2008 Wiley Periodicals, Inc. J Biomed
Mater Res Part B: Appl Biomater 88B: 558–578, 2009

Keywords: adhesion; collagen; demineralized; hydroxyl(1)apatite; molecular modeling

INTRODUCTION technique in dentistry in restorative procedures. For many

Adhesion to tooth has been a subject of considerable
research interest for several decades now. Because of poor
adhesion of restorative materials to prepared tooth, early
attempts to restore teeth emphasized surgical removal of
sound tissue by preparing the cavity to provide mechanical
retention through such features as dovetails, grooves,
undercuts, sharp internal angles and so forth. A major
advance in the promise of direct adhesion to tissues without
unnecessary sound tissue removal occurred with the discov-
ery by Buonocore1 that acid etching can significantly
enhance bonding of restorations to enamel. However, den-
tin did not yield comparable bonding through acid-etch
technique.2 As a result, etching of enamel became a routine
Correspondence to: T. K. Vaidyanathan (e-mail: vaidyatk@hotmail.com)
Contract grant sponsors: National Institute of Dental and Craniofacial Research,
National Institutes of Health; Contract grant number: 1 R01 DE14370
' 2008 Wiley Periodicals, Inc.
558
years, however, dentin-bonding procedures used a distinctly
different approach from that of bonding to enamel because
of the poor bonding on etched dentin surfaces as well as
major reservations about the danger of etching dentin surfa-
ces, which are connected to the pulp through dentinal
tubules. However, Fusayama3,4 challenged the assumption
that etching dentin does more harm than good, and force-
fully advocated the need for etching the tissue to improve
overall adhesion. According to Fusayama, it is far more im-
portant to seek adhesion to tooth through total etching (i.e.,
etching both enamel and dentin segments of the cavity
preparation) to promote bonding than to stick to the tradi-
tional box-form cavity preparation for promoting mechani-
cal retention.3 At about the same time, Nakabyashi et al.5,6
also demonstrated that the removal of mineral phase from
the surface layers of dentin by conditioning or acid etching
exposes the dentinal collagen matrix as a bonding substrate
(allowing adhesive infiltration), and that this is a safe and
practical approach to improve bonding to dentin. Because
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN
of the hydrophilic nature of this matrix, they suggested the
use of monomers with both hydrophilic and hydrophobic
groups for improved adhesion. The hydrophilic functional-
ity helps facilitate permeation of the monomer into the col-
lagen matrix leading to the formation of a collagen–resin
hybridized layer, whereas the hydrophobic functionality
facilitates bonding to the hydrophobic resin matrix in the
restoration. Such a procedure significantly improved bond-
ing and sealing at the dentin–restoration interface. The pro-
cess of hybridization is believed to result from the
infiltration of the primer into the open spatial network in
the collagen matrix exposed by dentin demineralization,
and its in situ polymerization. It soon became apparent that
the smear layer that forms on the tissue surface during cav-
ity preparation by mechanical tools was an important con-
sideration in the adhesion process. Conditioning (or
etching) the tissue removes any smear layer and may
potentially provide a predictable substrate for bonding.
Although different conditioning agents such as maleic, cit-
ric, phosphoric, and nitric acid at different concentrations
(e.g., 2.5% nitric acid, 10% maleic, and citric acids, 10–
37.5% phosphoric acid) have been tried as conditioning
agents, a 30–37.5% phosphoric acid is now apparently pre-
ferred because of its known advantage in efficiently etching
enamel to an operator identifiable frosted appearance
known for good adhesion to enamel and because of a well-
defined etching pattern for dentin. Several bonding systems
now use the total etching procedure because it is often con-
sidered as a gold standard for predictable adhesion to
tooth.7 Optimized etching procedures on enamel remove
the smear layer and typically create a honeycomb porosity
on its surface through preferential dissolution patterns of
the enamel prismatic structures. This facilitates the flow of
the adhesive into this porous substructure to form protrud-
ing anchor tags at the enamel-restoration interface for
improved mechanical bonding. Etching dentin removes the
smear layer and the hydroxyapatite (HAP) mineral phase
from the tissue surface, creating a network of exposed col-
lagen fibrils as the underlying substrate. Nakabayashi et al.
found that this was a very permeable layer facilitating the
infiltration of adhesives into the spatial network of the
fibrils.5,6,8 However, this substrate was soon identified as a
complex structure very sensitive to the ambient conditions
used. Kanca 9 observed that a slightly moist environment
during bonding improved the bond strength, and this proce-
dure became identified as wet bonding to dentin. The de-
mineralized collagen matrix of dentin was found to shrink
and collapse by drying,10 preventing adhesive permeation
into the substrate for efficient bonding. As a result of the
complex issues in dentin bonding, the physical changes
through demineralization of dentin and its adaptation to
ambient environment, and the morphological, structural and
physicochemical features of hybrid layer received extensive
research focus during the last three decades.5–26 Based on
the advances in understanding of dentin bonding as a result
of these studies, novel dentin bonding systems were also
Journal of Biomedical Materials Research Part B: Applied Biomaterials
559
developed that combined (a) total etching and rinsing (b)
priming using a bifunctional monomer with both hydropho-
bic and hydrophilic functionalities, and (c) bonding where
an adhesive comonomer was used that can efficiently bind
to both priming monomer and to the restorative resin
monomer. In the early stages of the development of dentin
bonding, the etching, priming, and bonding steps were car-
ried out separately. Subsequently, however, newer dentin
bonding agents were developed to combine one or more
steps of etching, priming, and bonding to improve operator
convenience. As a result, dentin bonding agents today fall
into three distinct groups: (1) Three-step systems, where
the etching (and rinsing), priming, and bonding steps are
carried out separately, All of these are total etch systems.
A commercial example of such a system is Scotch Bond
Multipurpose from 3M, (2) two-step systems which include
two different subtypes using (a) one total etching (and rins-
ing) step followed by application of a self-bonding primer
(e.g., Prime and Bond NT) and (b) application of a self-
etching primer followed by a bonding step (e.g., Clearfil
SE Bond from Kuraray), and (3) Single-step self-etching,
self-priming, self-bonding adhesives where the etching, pri-
ming, and bonding are combined into a single step (e.g.,
Adper Prompt L-Pop). These single-step systems are better
referred to as ‘‘all-in-one’’ systems to distinguish them
from other systems in which the manufacturers have used
the term ‘‘single bottle’’ and ‘‘one-step’’ to designate some
total etch formulations where only the priming and bonding
steps have been combined into a single step. Regardless of
the number of steps involved, however, the permeation and
interaction of the hydrophilic monomer within the porous
tissue substrate to form a hybridized tissue–resin hybrid
layer was found to be an important common feature in
effective dentin–adhesive bonding interface morphology.8
The primer and adhesive formulations typically contain a
mixture of resin monomers, and light, chemical, or dual
cure initiators and other additives. The priming agents con-
sist of at least one hydrophilic monomer, which is typically
also methacrylated for copolymerization with the adhesive
methacrylate monomers used in restorative applications. In
the self-etching primers, the hydrophilic monomers used
are also acidic to help etch the tissue. However, all-in-one
self-etching systems typically use a more acidic primer (pH

1) than that of two-step self-etching systems (pH 1.9–
2.4) where etching and priming only are combined into a
single step. In addition, the all-in-one adhesives are of
lower viscosity, which makes it difficult to keep it in place.
Despite the convenience of a single application step, all-in-
one adhesives have been reported to show less than opti-
mum performance in dentin bonding because of a variety
of problems associated with their higher hydrophilicity, low
viscosity, and monomer–solvent phase separation creating
water droplets in the adhesive after polymerization.27,28
Most priming agents interact with either the exposed
collagen fibrils (in total etch systems) or the intact dentin
tissue (in self etching systems) forming a hybrid layer.
560 VAIDYANATHAN AND VAIDYANATHAN

TABLE I. A List of Some Current and Promising micromechanical bonding was invoked by Nakabayashi
Hydrophilic Monomers et al.5,6 to explain the permeation of an adhesive monomer 4-
S. Short methacryloyloxyethyl trimellitate anhydride (4-META) into
No. Name Chemical Structure the porous collagen scaffold on the etched surface of dentin,
1 HEMA 2-Hydroxyethyl methacrylate and the encapsulation and entanglement of the collagen net-
2 GLUMA Adduct of glutaraldehyde and HEMA work by a polymer network formed by the polymerization of
3 NPG-GMA N-Phenyl glycine/glycidyl the infiltrated monomer. Although the permeation of the
methacrylate (adduct) monomer into the collagen scaffold should be viewed as a
4 NTG-GMA N-(p-Tolyl)glycine and glycidyl micromechanical phenomenon, atomic-level interactions
methacrylate potentially play a critical role in the overall adhesion process.
5 GDMA Glyceryl dimethacrylate It is well known that the triple helix motif in the collagen
6 GPDM Glycerophosphoric acid dimethacrylate structural hierarchy has morphological and compositional
7 NMBu N-Methacryloyl butyric acid
surface features favorable for ligand binding to promote a va-
8 NMGlu N-Methacryloyl glutamic acid
9 NAAsp N-Acryloyl aspartic acid
riety of biological functions.29,30 The triple helix motif in
10 NMHyp N-Methacryloyl hydroxyproline collagen structure is typically made up of three helical a-
11 NMGly N-Methacryloyl glycine chains of (Gly-X-Y) amino acids, respectively. Computer-
12 DIPENTA Pentaacryloyldipentaerythritol programmed collagen structural models incorporating the tri-
phosphoric acid ple helix motif visually demonstrate that the helical turns of
13 PMDM Diadduct of pyromellitic anhydride the triple helix create cavity tracks on the collagen molecular
with 2-hydroxyethyl methacrylate surface along the entire length of each collagen molecule.
14 MEM 2-Methacryloyloxyethyl hydrogen These tracks provide steric, hydrogen bonding, and electro-
maleate statically favorable interaction sites on the molecular surface.
15 MMEM Mono-2-(methacryloyloxy)ethyl
For example, the triple helix structure is known to have the
maleate
16 MMPM Mono-2-(methacryloyloxy)propyl hydroxyproline residues pointing outward from the molecu-
maleate lar surface, as seen in two projections of the currently
17 MDPM 3-Methacryloyloxypropyl phosphate accepted triple helix structure [see Figure 1(a,b)] with one
18 MBP 4-Methacryloyloxybutyl phosphoric interchain hydrogen bond per tripeptide.31 The sequence
acid shown is (Gly-Pro-Hyp)3, and each chain in the triple helix
19 MOP 8-Methacryoyloxyoctyl phosphoric has a different color ribbon drawn through the backbone. It is
acid believed that the structure is stabilized by hydrogen bonds
20 ACE Acryloyloxyethyl citraconate
between the backbone amide of a Gly residue and the back-
21 BMEP Bis[2-(methacryloyloxy)-
ethyl]phosphate bone carbonyl of X residue where the amino acid residue
22 EGMP Ethylene glycol methacrylate sequence is represented by a repeat of [-Gly-X-Y-]. The
phosphate backbone chains of adjacent molecules in each microfibril
23 MDP 10-Methacryloyldeacamethylene are believed bonded through hydrogen bonds between the
phosphoric acid hydroxyproline residues. Hydrogen-bonding interactions
24 PhenylP 2-Methacryloyloxy phenyl phosphate may also occur between side chains with other residues.
25 4-META 4-Methacryloxyethyl trimellitate Some hydroxyproline residues of the triple helix molecules,
anhydride
and other backbone or side chain hydrogen-bonding moieties
26 PA 2-Acryloyloxyethyl phosphate
27 PM 2-Methacryloyloxy ethyl phosphate
are still available for additional hydrogen bonds, and a hydra-
28 SBMA 3-Sulfo-2-butyl methacrylate tion network connecting water and these hydroxyproline resi-
29 SEMA 2-Sulfoethyl methacrylate dues has been demonstrated in the native and wet states of
30 MSPMA 3-Methoxy-1-sulfo-2-propyl collagen.30 This is considered important in the case of wet
methacrylate dentin bonding technique (where the priming step is carried
out under moist conditions) because it improved the bond
strength vis-a-vis dry bonding technique where the cavity
They are, therefore, critical ingredients in the formulations surfaces were completely dried before priming.14–17 In wet
that promote tissue–adhesive interactions. A selected list of bonding, the primer monomer is infiltrated into the hydration
currently used or potential hydrophilic monomers that can be network of the collagen scaffold, and when the water of
considered as priming agents is given in Table I. Because of hydration is removed, hydrogen-bonding, van der Waals, and
the importance of priming step in dentin bonding, the mor- electrostatic interactions between the primer and the collagen
phological and analytical aspects of primer–tissue interaction molecules may be facilitated. Both monomer and its carrier
have been explored extensively. However, the nature of these solvent molecules in a wet bonding primer formulation
interactions remains uncertain and not fully understood. should be compatible with the hydration environment of the
There was a consensus until recently that the interactions are collagen scaffold. Typically therefore monomers incorporat-
micromechanical rather than physicochemical. The nature of ing a sufficiently hydrophilic functionality (e.g., acidic,

Journal of Biomedical Materials Research Part B: Applied Biomaterials

 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN 561

Figure 1. Ball and stick diagrams showing two projections longitudinal (a) and cross-sectional (b)
views of currently accepted triple helical structure of collagen. One interchain hydrogen bond per
tripeptide is assumed. The sequence shown is (GLY-PRO-HYP)3 and each chain in the triple helix
has a different color ribbon drawn through the backbone. The projections elegantly illustrate how
the hydroxyproline residues point outwards from the triple helix for facilitating hydrogen bonding
between collagen molecules in the microfibril and between the microfibrils and other hydrogen-
bonding molecules like water or other ligands. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]

hydroxyl, amide etc.), formulated in water-compatible sol-
vents (e.g., water, ethanol, or acetone), are used as primers to
facilitate the formation of an effective hybridized layer.
Methacrylate functionality is also incorporated into the
monomer to facilitate its homopolymerization and its copoly-
merization with methacrylate bonding agents (referred to as
adhesive monomer) used as a bridge to bond to resin-based
composite and other restorations.
As pointed out earlier, unlike the total etch adhesive sys-
tems, two-step self-etching or one step all-in-one bonding
systems use acidic primer monomers. These monomers are
believed to chemically interact with the HAP phase of dentin
through charge interactions.32 In addition, interactions of the
primer monomer may also occur with the collagen matrix
phase. The current trends in clarifying these collagen–primer
and HAP–primer interactions need further examination.
PRIMER–TISSUE INTERACTIONS
Total Etch Adhesives
A wide range of bond strengths has been reported in the
literature for three- and two-step adhesives classified as
total etch systems (33–38). Although bond strength differences

Journal of Biomedical Materials Research Part B: Applied Biomaterials

562 VAIDYANATHAN AND VAIDYANATHAN
can be associated with many interactive factors, such as, the
solvent used, additives, the etchant used, variability in tooth
structure, incomplete permeation into the demineralized layer
of dentin, and other factors; monomer chemical differences
are considered important in monomer–tissue interactions after
effective permeation. Two approaches have been pursued to
understand the role of adhesive–primer monomer interactions
with demineralized dentin matrix in dentin bonding. They
are: (a) analysis of solubility parameter compatibility between
the adhesive and collagen substrate and (b) direct/Indirect
computer modeling of collagen–ligand interactions. A brief
overview of these approaches in the current state of the art
are presented later.
Application of Solubility Parameter to Dentin Bonding
Rationale and Basic Concepts. The solubility parame-
ter concept has been used to explain the role of adhesives
in dentin bonding, and the basis of such application is
briefly reviewed here. As early as 1936, Hildebrand pro-
posed a theory to explain miscibility between two nonelec-
trolyte liquids.39 He used the ‘‘like dissolves like’’
principle, and suggested that a liquid will dissolve in
another when both liquids have similar cohesive energy
density (CED). The cohesive energy of a liquid is calcu-
lated from the heat of vaporization (DEv), representing the
molecular attraction between molecules in the condensed
state of the material. Hildebrand proposed the use of the
square root of the CED (defined as cohesive energy per
unit volume, i.e., CED 5 DEv/V, where DEvi is the energy
of vaporization for a given material ‘‘i’’ and Vi its volume)
as a numerical index of the solvency behavior of any liq-
uid, and designated it as solubility parameter d. According
to Hilderbrand concept, the distribution of d values of
liquids, defined by H(DEv/V) represent a spectrum of sol-
vency behavior of liquids, in which mutually miscible
liquids may have comparable d values. The theory has seen
significant modifications and refinements by several authors
(notably 40–52), including its expanded application to liquid
electrolytes and adhesive coatings to polymer or other sub-
strates. Burrel40 suggested that some of the inconsistencies
in the application of Hilderbrand’s solubility parameter to
specific liquids can be overcome by incorporating the role
of hydrogen bonding into solubility consideration by divid-
ing solvents into three groups (a) weak hydrogen-bonding
liquids (e.g., hydrocarbons, chlorinated hydrocarbons, and
nitrohydrocarbons), (b) moderate hydrogen-bonding liquids
(e.g., ketones, esters, ethers etc.), and (c) strong hydrogen-
bonding liquids (e.g., alcohols, amines, acids, amides, and
aldehydes). It soon became apparent that the Hilderbrand
solubility parameter could also be extended to liquids other
than nonpolar reagents such as polar solvents with appro-
priate modification. Hansen and Skaarup41–43 expanded the
solubility parameter concept by incorporating three contri-
butions to solubility parameter derived from CED, namely
a dispersion contribution (dd) to represent van der Waals
interaction forces, a polar contribution (dp) due to charge
interactions and a hydrogen-bonding contribution (dh). If
the three contributions are represented in Cartesian coordi-
nates, the overall solubility parameter dt can then be
regarded as a vector in three-dimensional (3D) coordinate
space, and can therefore be defined as: d2t 5 d2d 1 d2p 1 d2h .
Hansen also developed methods for the 3D representation
of these three contributions for individual liquids, and to
use such 3D representation to characterize their miscibility
from the positions of pairs of liquid media in the 3D space.
Hansen showed that by plotting the solubility data of a
polymer dd, dp, and dh in 3D coordinates, the upper limits
of these parameters for experimentally observed miscibility
range of a polymer in solvents of different solubility pa-
rameter values can be used as a radius for a sphere of solu-
bility with the solubility parameter value of the polymer
itself as its center. A spherical volume of solubility can
thus be formed for each polymer. Thus, the radius of a
sphere of solubility [D(A 2 B)] between the components A
and B in this 3D (dd, dp, dh) space is calculated from the
relation: [D(A 2 B)]2 5 [dd(A) 2 dd(B)]2 1 [dp(A) 2
dp(B)]2 1 [dh(A) 2 dh(B)]2. Typically, miscibility is
observed for [D(A 2 B)] values of \5 MPa1/2. Thus, if a
sphere with a radius of 5 MPa1/2 is constructed in the
above 3D space, solution mixtures with solubility parame-
ters that fit within that space are miscible with each other,
but those that fall outside the sphere are not. Such a refer-
ence sphere can be used to select active solvents for use
with a given polymer. Two dimensional models of the sol-
ubility sphere (e.g., Hansen’s dd 2 dh plots and Bagley’s
dv 2 dh plots44 where dv 5 dd 1 dp) are also useful to
illustrate changes in solubility parameter contributions and
their effect on miscibility between solvents and between a
solvent and a polymer. Teas45 also suggested the use of a
single plane graphical method to represent the three solu-
bility contributions on three sides of an equilateral triangle
as fractional solubility parameters fd, fp, and fh derived
from dd, dp, and dh as defined below:
dd dp
fd ¼ ; fp ¼ ;
dd þ dp þ dh dd þ dp þ dh
dh
fh ¼
dd þ dp þ dh
where, fd 1 fp 1 fh 5 1 5 overall solubility parameter.
In this approach, the overall solubility parameter is fixed
arbitrarily at 1 (or 100, if expressed in percent ratios of the
individual contributions) for all chemicals, and fractional
solubility parameters fd, fp, and fh are expressed as applica-
ble contributions to it due to dispersion, polar, and hydrogen
bonding based on their chemical make-up [as illustrated in
Figure 2(a) representing a solubility parameter value made
up of fd, fp, and fh in the ratio 50:35:15]. According to this
assumption, solubility behavior is determined not by differen-
ces in total Hildebrand value, but by the relative amounts of
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN 563

Figure 2. Teas diagrams representing fractional solubility parameter, (a) method of fractional solu-
bility parameter representation and (b) positions of typical solvent classes in the Teas diagram rep-
resentation.

the three component forces that contribute to the total Hilde-
brand value. Figure 2(b) shows a Teas graph for some com-
mon classes of solvents. One advantage of such
representation is the ease with which the nature of the sol-
vent characteristic can be deduced from the location of the
solvent in the graphical plot. Thus, aliphatics and aromatics
are recognized to possess dd as the dominant contribution to
their overall solubility parameter, dh is the dominant contri-
bution to glycols, dp is the dominant contribution to nitro
compounds and so forth. Thus, although the theoretical basis
of Teas graph is not clear, it is a very convenient method to
visualize solubility parameter make-up of individual chemi-
cals relative to other chemicals of interest.
Using cohesive energy as an additive property for com-
mon small molecules, many authors (e.g.,47–52) developed
group contribution models for solubility parameter calcula-
tions using atom groups in molecules. Small48 defined molar
attraction constant Fi 5 DEvi Vi for any individual chemical
group i in a molecular structure and suggested that it can be
used as an additive property for different chemical groups to
calculate the overall solubility parameter of organic com-
pounds. Thus, if a molecular structure contains multiple
chemical groups, the overall solubility parameter is given by
dt 5 (SFi/SVi). Using Small’s concept of molar attraction
constants separately for both dispersion and polar contribu-
tions, van Krevelen and Hoftzyer50 developed model equa-
tions for group contributions to dd, dp, and dh to arrive at
overall solubility parameter d as follows:
qffiffiffiffiffiffiffiffiffiffiffiffi
P 2 sP
ffiffiffiffiffiffiffiffiffiffiffiffi
P Fpi
Fdi Ehi
dd ¼ P ; dp ¼ P ; dh ¼ P
Vi Vi Vi
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
and d ¼ d2d þ d2p þ d2h
In the above equations, Fd is the dispersion molar attrac-
tion constant, Fp is the polar group molar attraction con-
stant, Eh is the hydrogen bonding contribution to the
cohesive energy, and i is a contributing group. Hoy51,52
subsequently developed an alternate group contribution
method involving semiempirical calculations to determine
the nonpolar, polar, and hydrogen-bonding parameters to
the total solubility parameter. He calculated the total solu-
bility parameter (dt) and the polar (dp), and hydrogen-bond-
ing (dh) contributions to it by individual chemical groups,
and derived the group dd values by difference method using
the relation d2t 5 d2d 1 d2p 1 d2h . Such group contribution
calculation methods are now considered to be remarkably
accurate. In addition to these methods to determine the sol-
ubility parameters of individual molecular components, it
has also been demonstrated that solubility parameter of a
mixture of more than one component can also be calculated
by incorporating the volume-wise contributions of the solu-
bility parameters of the individual components of the mix-
ture. The fractional parameters for each component are
multiplied by the fraction that it occupies in the blend, and
the results for each parameter are added together. In this
way, the position of the solvent mixture can be located
graphically on appropriate 3D or two-dimensional (2D)
plots and Teas plots. The group contribution method is a
very convenient approach to calculate the solubility param-
eters of individual chemicals in a mixture and the cumula-
tive solubility parameter of a mixture of multiple
chemicals. The method is now available in user-friendly
commercial computer programs, and an application soft-
ware using Hoy’s group contribution method (Computer
Chemistry Consultancy, Seningen, Germany) is now exten-
sively used by several researchers, including many pub-
lished articles in dentin bonding as discussed later.
Application of Hoy Parameters to Dentin Bonding
It is thus apparent that when the solubility parameter value
(or its dominant contribution) of an adhesive matches (or is

Journal of Biomedical Materials Research Part B: Applied Biomaterials

564 VAIDYANATHAN AND VAIDYANATHAN

close to) the corresponding solubility parameter value of a

polymer substrate, the adhesive is expected to interact with
the substrate. Such interaction has been shown to cause
swelling and permeability changes in many polymers, as
described in a recent review on polymer dissolution.53 The
application of Hoy’s solubility parameter contribution
method and its relationships to interactions between dentin
adhesives and demineralized dentin have recently been
studied in several published articles.54–59 After Asmussen
et al.54,55 and Miller et al.56 demonstrated the potential use
of solubility parameter in dentin bonding systems, Chappe-
low et al.57 have shown that shear bond strengths of iso-
cyanatomethacrylate dental adhesives to etched dentin
surfaces revealed a strong correlation with approximate
permeability values (P0 ) of different adhesives calculated
using the relation P0 5 (Ddt)21/2 M21/2, where P0 is a Figure 3. A potential approach to modify the Hoy’s solubility pa-
rameter contributions by choice of solvents to make the adhesive
modified permeability (incorporating a proportionality con-
and the collagen matrix compatible for permeability. By dissolving
stant for other potential variable contributions to the normal the adhesive BisGMA in ethanol, BisGMA solubility parameters are
permeability), Ddt is the mismatch in the total solubility pa- shifted to fall within the solubility sphere of collagen which in turn
rameter between etched dentin substrate and the adhesive, shifted by replacing the ambient water environment in the collagen
and M is the weight average molecular weight, a factor matrix by ethanol.
that controls diffusion coefficient of the permeating species.
Similarly, King et al. showed that in blended copolymer
This approach is therefore extremely valuable to under-
films of increasing hydrophilicity, the permeability of the
stand the permeation of the adhesive into the collagen
film follows a linear correlation to the total solubility pa-
fibrillar network. However, increased swelling-induced per-
rameter of the monomer blend.58 Other reports have related
meation typically can be expected to increase the depth of
the swelling and other effects of solvents on demineralized
permeation and consequently the thickness of the hybrid
dentin to differences in solubility parameters between the
layer and this should lead to an increase bond strength
solvent and the substrate.59–62 In the case of a demineral-
under a purely micromechanical model. However, there is
ized dentin, swelling of the collagen matrix can promote
no evidence that increased hybrid layer thickness contrib-
permeation of the ligand to the substrate. It is hypothesized
utes to improved bond strength or adhesion.34,65 In addi-
that permeation of a hydrophilic monomer into the swollen
tion, other complicating factors may interfere with effective
collagen matrix can lead to an efficient hybridized layer by
monomer permeation. For example, collagen triple helix
encapsulation and entanglement of primer polymer chains
has a tight internal structure. Moreover, even if complete
after polymerization within the collagen scaffold. Recently
demineralization of dentin occurs during conditioning,
Tay et al.63 and Sadek et al.,64 have shown that the bond
water will still be present in collagen intrafibrillar space in
strength of hydrophobic adhesive systems may be related
wet bonding as a water-bridged hydration network. This
to controlled ambient changes in the demineralized dentin
intrafibrillar water is in a bound state through hydrogen
substrate environment. By replacing the water in collagen
bonds. X-ray diffraction studies on the structure of
intrafibrillar spaces with ethanol, they found that the bond
hydrated collagen have shown that even when the extrafi-
strength of hydrophobic resins in ethanol solvent to demin-
brillar water is frozen by lowering the temperature to 08C,
eralized dentin could be increased significantly. They
the intrafibrillar water remains bound without freezing.66
related this change in bond strength to the increase of the
solubility parameter dh in neat BisGMA toward higher val- This indicates the difficulty for primer monomer systems to
ues by dissolving it in ethanol and the decrease in both dh compete with and displace water in the intrafibrillar space.
and dp of the collagen matrix by the replacement of its am- This would suggest that penetration of monomer into the
bient water environment with ethanol. These shifts bring intrafibrillar spaces is somewhat restricted, and encapsula-
the BisGMA monomer in ethanol toward the solubility tion and entanglement may primarily occur in extrafibrillar
sphere of collagen matrix in ethanol, as seen in Figure 3. spaces, and cause less than optimum overall adhesion often
The effect of solubility parameter differences is thus poten- considered responsible for the micromechanical model. The
tially an important avenue to characterize the ability of a encapsulation/entanglement effects are critical for increased
primer monomer system to infiltrate into the collagen scaf- bond strength in a purely micromechanical model. At the
fold. The major advantage of this method is that the calcu- same time, permeation can occur efficiently into the extrafi-
lations can be used to characterize not only single brillar space. When the adhesive atoms approach atoms on
molecular adhesive species but also multicomponent pri- the exposed collagen molecular surfaces in the extra- or
ming mixtures using additive relations involving multiple intrafibrillar space, atom–atom interactions between the ad-
components. hesive and the collagen substrate may be significantly

Journal of Biomedical Materials Research Part B: Applied Biomaterials

 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN
enhanced. When this water is replaced by a hydrophilic
monomer during dentin bonding, the monomer can be
expected to interact with the collagen. It is therefore rea-
sonable to suggest that nonbonded, electrostatic, and hydro-
gen-bonding forces between the monomer phase and the
collagen molecules may develop after permeation. Some
direct analytical evidence for hydrogen-bonding or other
interactions have also been previously reported in the liter-
ature.67–69 Such interactions are also evident from the role
of different Hoy’s solubility parameters dd, dh, and dp
invoked in recent published research in dentin bonding.63,64
The overall bonding should, therefore, be viewed as inclu-
sive of adhesive energy associated with these interactions,
in addition to the encapsulation and entanglement effects
after polymerization of the infiltrated monomer. Under
these conditions, a thorough understanding of the ligand–
collagen interactions is also essential to fully characterize
dentinal adhesion through etching and subsequent bonding.
In analyzing the intermolecular interactions between primer
monomers and collagen molecules, it is to be borne in
mind that there is no evidence of any primary bond
between collagen and the monomer molecules. The interac-
tion is therefore primarily due to secondary bonds including
van der Waals interactions together with limited electro-
static and hydrogen-bonding interactions. The energy of
such secondary bonds is typically of the order of a few
kcals/mole (1–7 kcals/mole), as opposed to much higher
values for primary bonds (e.g., 50 kcals/mole or much
higher for ionic or covalent bonds). Many computer pro-
grams are now available to simulate these interactions and
calculate the interaction energies. The second approach
used to characterize the bonding of dentin adhesives with
collagen matrix invokes the role of collagen–ligand–adhe-
sive interactions, using computer modeling and immuno-
chemical binding assays.
Computer Modeling Analysis of
Collagen-Adhesive Interactions
Over the past few decades, there have been revolutionary
advances in the development of computational models to
virtually depict simple and complex molecules as 3D mod-
els in a computerized database. Such models incorporate
the atomic coordinate positions (which define bond lengths
and bond angles in the individual molecules), charge distri-
butions, and other features into the database that can be
readily visualized as a 3D structural model in a computer
screen. Additionally, these computational models can be
used to study interactions between molecules (assuming
different conformational states) using sophisticated software
programs, and the outcomes of simulated interactions can
also be visualized with energetically favored geometric
relationships between the different molecules in the inter-
molecular complex that is formed. These methods make it
possible to probe at the atomic-level biochemical interac-
tions that occur at localized sites of large target molecules
Journal of Biomedical Materials Research Part B: Applied Biomaterials
565
due to the presence of other reactive molecules in their
close vicinity.
Recent computer simulation studies have shown that
ligand binding to a type 1 collagen can be assessed compu-
tationally through molecular modeling.70–75 Early studies
were carried out with an initial collagen structural model in
the RCSB PDB file 1CGL.76 In recent years, more accurate
collagen structural models established by crystal structure
studies have become available as newer RCSB PDB files
(see an available list by Rainey and Goh).77 In addition,
several computer programs70 have also become available to
study intermolecular interactions between collagen as target
molecule and ligands as adhesives. An important advantage
of the computer simulations is that the interactions are
visualized with 3D structural models that can mimic rapid
energy fluctuations between different stable/metastable
states reflecting conformational changes of the adhesive
molecules. Consequently the results reflect the effect of
molecular flexibility that may lead to multiple binding pro-
files in a complex target structure such as collagen. Two
distinct approaches are available to characterize these inter-
actions: target-based docking methods and ligand-based
analysis of common functional alignment patterns.78–87 In
the target based docking method, the interaction energy is
computed using three different components of interaction
between adhesive molecules in different conformational
states and rigid target molecules, na;mely: (a) van der
Waals interactions; (b) electrostatic interactions; and (c)
hydrogen bonding. In the ligand-based approach, the low
energy conformational space of the ligand molecule is
explored and common patterns of their important functional
alignments in the conformational landscape are treated as
their favored binding poses. A brief description of these
approaches is discussed later.
Target-Based Direct Methods: Docking Simulations
Modeling of ligand–protein interaction has attracted much
research interest in the past few decades.78–84 Typically
when interactions between biomolecular target species and
other ligand molecules are considered, the degrees of free-
dom such as conformational, translational, and orientational
flexibility of both the target and the ligand structures
should be taken into account, and this makes the calcula-
tions quite complex. However, a giant macromolecular tar-
get such as collagen (typically anchored to intact dentin
and or hydrogen bonded to the water of hydration in the
native state) can reasonably be considered as a rigid and
static target with no conformational, translational, or rota-
tional freedom. On the other hand, ligands used to prime
the demineralized dentin are small molecules typically in
liquid state and possess the translational, orientational, and
conformational freedom. In fact, exclusive computer pro-
grams have been developed to model interactions between
such a large ‘‘rigid’’ biomolecular target and small
‘‘flexible’’ molecular species such as the primer molecules
566 VAIDYANATHAN AND VAIDYANATHAN
used in dentin bonding.78,83,84 AutoDock (Scripp Research
institute) is a popular program, which has been used to
study collagen–ligand interactions.83,84 In this program,
computer simulations help locate the favored target site(s)
where the ligand tends to preferably dock due to energy
minimization through increased interaction. Such a simula-
tion also develops a matrix of ligand–target interaction
energy (or binding energy) values for ligand conformations
docked to the target at all favorable sites around a rigid
and static target.70 This is accomplished in AutoDock by
precalculating the van der Waals, hydrogen-bonding and
electrostatic contributions to the total interaction energy
values by appropriate calculations based on atom probes or
their charges placed on each discrete point of a grid sur-
rounding the target region of interest, and using these val-
ues to determine the interaction energy at any point of
probe or charge location by interpolation. In AutoDock,
these calculations are carried out with an implicit solvent
(water) model, making it valuable for use in collagen–
ligand interaction studies.70,72 The overall interaction
between molecules is typically broken down to a summa-
tion of atom–atom dispersion or van der Waals interactions,
charge interactions, and hydrogen-bonding interactions
using interatomic distances and coulombic charges
involved. The interactions are modeled through Lennard
Jones 12-6 potential energy function for van der Waals
interactions [given by (Aij/r12ij 2 Bij/rij ), where Aij is the re-
6
pulsive term coefficient and Bij the attractive term coeffi-
cient for atoms i, j separated by distance rij] and through
the 12-10 potential energy function for hydrogen bonding
ij 2 Dij/rij ), where Cij and Dij are repulsive
[given by (Cij/r12 10
and attractive term coefficients, respectively, and rij is the
distance between atoms as before]. The electrostatic energy
due to Coulombic interaction between two atoms ‘‘i’’ and
‘‘j’’ is given (in a simple model) by Eele 5 (qiqj/4pe0rij),
where qi, qj are atomic charges of interacting atoms i and j;
e is the dielectric constant, and rij is the distance between
the charges. The total interaction energy is the sum of 12-6
and 12-10 potential energies and electrostatic energies of
interaction between each target atom of interest and all
ligand atoms involved. These values are calculated for a
vacuum environment, and adjusted for the screening effect
of water in the environment. The positive free energy of
the ligands due to torsional freedom at flexible single bonds
is also subtracted from the overall interaction energy. To
localize the interaction energy calculations to a set of target
sites, the calculations are confined to a 3D grid box of
proper dimensions to cover all target sites of interest. Such
molecular modeling methods are very valuable computa-
tional tools to study collagen–ligand interactions quantita-
tively, as has been demonstrated in several previous
research reports. Because these methods predict intermolec-
ular interactions through theoretical studies, in vitro meth-
ods are needed to support or supplement these studies. For
example, binding assay methods of assessing changes in
antibody binding as a result of prior exposure and binding
of ligands to a target structure is a potentially valuable ex-
perimental approach to study collagen–ligand interactions.
Microstructurally, the nature and characteristics of primer
permeation into the exposed collagen matrix and the nature
of the interphases (e.g., hybrid layer and the adhesive resin
phase) connecting the intact tissue to the restoration may
be important in analyzing the effectiveness of priming effi-
cacy. Such studies have been reported in the past, but more
robust correlations of these theoretically calculated interac-
tion energy values to clinically important properties, such
as bond strength, are needed in the future.
Vaidyanathan et al.,70–75 have recently reported com-
puter modeling studies involving automatic docking of
ligands on to a type 1 collagen structure. Typically dock-
ings of ligands to the target were at the cavity tracks of
the collagen molecule with functional complementarity
(electrostatic/hydrogen bonding) and/or steric complemen-
tarity. Interaction energy optimization was typically the
result of bringing the favored conformations to closer
distances of atom groups and functional attributes of the
target and the ligand. The results can be briefly summar-
ized as below:
1. The effect of primer formulations is predicted to be
through collagen–ligand interactions. Such interactions
may result when specific features (functional groups
and spatial features) in ligands in low-energy (stable)
conformational states are configured in alignment with
complementary features in the collagen target. Thus
charge, hydrogen bonding, and spatial alignment
between the target and the ligand may result in electro-
static, hydrogen bonding, and steric complementarity,
as illustrated in Figure 4. Visualization of bound con-
formational poses typically show that one or more fea-
tures of the ligand in their lowest energy states are
properly aligned to the collagen target features to mini-
mize the overall energy of the target–ligand complex.
The overall binding energy values are negative indicat-
ing that the interactions are thermodynamically favored
to occur; however, the lowest calculated binding
energy values are typically of the order of 25 kcals/
mole, as is expected from the lack of charge contrast
between the ligand and the target. Figure 5(a) also
shows that interactions are favored at sites along heli-
cal tracks associated with the triple helix, because
these tracks typically represent sites with steric, elec-
trostatic and hydrogen-bonding complementarity to the
ligand conformational poses that dock at these sites.
2. Significant differences in interaction energy were esti-
mated due to differences in composition, molecular
size and functional groups of the ligand structure.73,74
Although the van der Waals interactions are predomi-
nant, hydrogen-bonding and electrostatic interactions
are also found between collagen and the ligand. Hydro-
gen bonds were often visualized as illustrated in Figure
5(b). These hydrogen bonds were typically between
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN 567

Figure 4. Illustrations showing steric, electrostatic, and hydrogen-bonding complementarity that

facilitates energy minimized docking of ligands. Ligand molecules are identified as ‘ b’’ in all cases.
(a) Steric compatibility between the cavity shape and a ligand molecular conformational pose that
facilitates closer access of the ligand into the cavity. (b) Electrostatic compatibility where the posi-
tively (color coded red) and negatively (color coded purple) charged localized sites in ligand seem
to approach the oppositely (negatively and positively, respectively) charged sites in the collagen tar-
get during docking. (c) Hydrogen-bonding compatibility where the hydrogen bond donor site (color
coded red) in the ligand is approaching hydrogen bond acceptor site (color coded blue) during
docking. [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]

hydroxyproline projecting outward from the collagen
triple helix and the amide I and amide II present in
type 1 collagen, with the hydroxyl and acidic (carbox-
ylic/phosphoric acid) groups present in the ligand.
3. Limited changes in the collagen structure have no sig-
nificant effect on the calculated interaction energy val-
ues, except in the relative contributions of van der
Waals and electrostatic energies to the overall binding
energy.73,74 Docking simulations involving collagen
target with minor modifications in composition [e.g., a
single (Gly-Glu-Lys) or (Ala-Pro-Hyp) triplet in the
middle of 10 triplets of the (Gly-Pro-Hyp) model
sequence] resulted in statistically significant, but only
small differences in the van der Waals and electrostatic
interaction contributions to the overall docking/binding
energy values.73,74 These differences seem to result
from changes in spatial locations of atoms and charge
distributions in the collagen target, leading to electro-
static/steric complementarity differences between the
target and the ligand.
4. Interactions between HAP phase and the adhesive may
potentially influence the priming of collagen target
because ligands dissolve calcium, generating cationic
organic ligands, which may also interact with the tar-
get.73 The cationic charges seem to increase electro-
static interaction between ligand and collagen when
collagen composition includes negative charge centers
such as glutamic acid residue.
5. The conformational flexibility of the ligand plays a
critical role in the overall binding process.74 Thus,
when torsional freedom facilitates flexible alignment of
the functional groups and features to complementary
sites in the target, the torsional freedom also introduces
a positive-torsional free-energy contribution to the
overall binding energy. Thus an optimum level of con-
formational flexibility through spacer units (such as
methylene groups) may help improve overall binding,
and this can be approximately theoretically determined
by binding energy estimates.
6. There are many similar binding targe sites for the
ligand in the collagen structure because of the repeat
sequence of amino acid residue triplets, and this may
result in interactions at multiple sites on the target.74
However, there is a window of energy range where the
binding conformations are energetically favored. There-
fore, overall adhesion of ligand to collagen may result
from multiple conformations bound to their respective
favored target sites within an energy range window.
Based on this, a model can be developed for the over-
all adhesion process from expected individual confor-
mational energy diversity of each ligand.70 This model
is based on the Boltzmann distribution of the ligand
conformational states. The Boltzman distribution stipu-
lates that the probability of the occurrence of a confor-
mational state pi is proportional to its energy by the
relation: pi 5 Ke2Ei/RT, where K is a constant, Ei is
the energy of a given conformation, R is the universal
gas constant, and T is the temperature. If we consider
two conformational states p1 and p2 with energy values
E1 and E2, the above relationship leads us to the rela-

Journal of Biomedical Materials Research Part B: Applied Biomaterials

568 VAIDYANATHAN AND VAIDYANATHAN
Figure 5. Visualization of typical docked ligand conformations on
collagen target obtained by AutoDock illustrates how the ligand
molecules align to the collagen cavity tracks due to binding interac-
tions promoted at these sites, and may establish hydrogen bonding
at specific target sites. (a) Docked conformations along the cavity
tracks. Space filling model to illustrate alignment of ligand along
cavity tracks on collagen triple helix. (b) Docked conformation illus-
trating hydrogen bonding (marked as dashed lines between collagen
target and ligand). Ball and stick model to illustrate the bonds.
[Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
tive ratio of their occurrence, that is, p1/p2 5 (E2 2
E1)/RT. Because the lower energy states are thermody-
namically favored to be stable, we can determine the
fraction of the number of higher energy states to that
of the lowest energy state by the above relation. For
example, the ratio of the probability of occurrence of
any given energy state to that at the minimum energy
state in the ensemble of stable conformations can be
calculated. Using the above relation at body tempera-
ture (378C, i.e., 3108K), the probability of a stable con-
formational states 3 kcals above the lowest energy
state (i.e., DE 5 3 kcals) can be shown to be \0.0077.
Thus it is clear that there is more than 99% chance of
stable conformational states being restricted to an
energy window between a lower bound of the mini-
mum energy state and an upper bound of 3 kcals above
the minimum energy state. Thus, although there are
potentially a continuum of conformational states possi-
ble, we need to consider only a relatively narrow
energy window of favored stable and/or metastable
conformational states to characterize the adhesive prop-
erties of any ligand. This is because the aggregate
properties of a molecular structure are predominantly
determined by the conformational states in the lower
range of their spectrum of energy values, where they
are most stable. Typically 50 conformational states
covered the above 3 kcal energy range in our calcula-
tions, and thus, adhesive characteristics of the ligands
were assessed to a first order of approximation by the
range and mean values of the 50 lowest energy docked
conformations. Our results showed that the energy
range and means of bound conformations differed sig-
nificantly (p \ 0.05) between different ligands, and
these differences were related to the functional or fea-
ture differences among the ligands studied.
Thus direct docking simulations provide valuable infor-
mation on the potential for atomic-level interactions
between collagen and adhesive ligands. These types of
interactions (van der Waals, hydrogen bonding, and electro-
static) are also the basis of the Hoy’s solubility parameter
concepts guiding the cohesive energy compatibility
between demineralized dentin and hydrophilic monomers.
Thus the two approaches are valuable, complementary tools
to understand the overall adhesion process involving hydro-
philic monomers and demineralized dentin. We have used
immunochemical binding assay methods to analyze the
ability of typical adhesive monomers to bind to collagen.
The monomers studied were (a) 10-methacryloloxy deca-
methylene phosphoric acid (MDP), (b) N-methacryloyl glu-
tamic acid (NMGlu), (c) Monomethacryloyloxyethyl
maleate (MMEM), (d) N-methacryloyl hydroxyproline
(NMHyp), and (e) N-acryloyl aspartic acid (NAAsp). The
procedure involved 1-h exposure of demineralized dentin to
water and ligands, respectively, as control and experimental
groups, followed by additional exposure of both groups to
anticollagen to facilitate antibody binding to collagen target
molecules. The samples were then treated to nanogold-la-
beled secondary antibody (15-nm nanogold labels) to bind
to anticollagen. The bound secondary antibody gold labels
were silver enhanced using a silver enhancement kit, and
the enhanced particles were visualized by secondary elec-
tron imaging using a Hitachi S-4800 field emission scan-
ning electron microscope (SEM). Figure 6 show typical
SEM images of control samples and experimental samples
treated with different ligands. The results reveal differences
in binding profiles as expected from the calculated binding
energy differences. However, all ligands bonded to dentin
sections were also examined by SEM and showed similar
interfacial hybridization (Figure 7) at the dentin–adhesive
interface, indicating that interaction energies may differ
even when permeation profiles may be similar.
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN 569

Figure 6. SEM images of silver-enhanced gold label binding profiles of acid-conditioned dentin sur-
face after ligand treatment binding assay. The figures illustrate how the different ligands tend to
bind to the demineralized dentin, causing a reduction or elimination of antibody binding expressed
by gold labels associated with secondary antibodies. A control treated only with water and no
ligand treatment is used as the reference for maximum antibody binding expression. Ligands used
are: (a) 10-methacryloloxy decamethylene phosphoric acid (MDP), (b) N-methacryloyl glutamic acid
(NMGlu), (c) Monomethacryloyloxyethyl maleate (MMEM), (d) N-methacryloyl hydroxyproline
(NMHyp), (e) N-acryloyl aspartic acid (NAAsp), and (f) control. All images were at original 10,0003
magnification.

Indirect Ligand Based Binding Poses. One of the basic
premises of computational approaches to biological prob-
lems is that biological activity is dependent on the 3D
placement of specific functional groups in molecules that
facilitates favorable interaction to a target molecular
site(s).70–72 Therefore analysis of common patterns of 3D
placements of chemical functions in likely conformational
states of all ligands known to bind to a target may reveal
potential binding poses of functional groups of ligands to
that target. Each pattern is called a pharmacaphoric hypoth-
esis,85 representing a common 3D spatial pattern of speci-
fied types of functions (e.g., hydrogen-bonding, acidic,
polymerizable functionality), which are present in the
ligand structures. If multiple binding patterns of poses are
presented by the ligands, these poses may represent alter-
nate binding models. The HypoGen and HipHop are two
modules in Catalyst software from Accelrys,86,87 that are
examples of such computer programs for pharmacaphore
search. Although HypoGen is a more comprehensive tool
to characterize the ligands based on their actual binding ac-
tivity profiles, detailed activity data of the dentin bonding
ligands are not presently available for use of HypoGen.
HipHop module focuses on commonality of functional ori-
entations of ligand conformations to identify pharmaca-
phore models. Typically, well-differentiated stable
conformational states of a set of ligands known to bind to
a specific target are searched and identified, and the com-
mon pattern(s) of their functional placement in 3D space

Journal of Biomedical Materials Research Part B: Applied Biomaterials

570 VAIDYANATHAN AND VAIDYANATHAN

Figure 7. SEM images of dentin–adhesive interfaces bonded with different ligands. (a) 10-metha-
cryloloxy decamethylene phosphoric acid (MDP), (b) N-methacryloyl glutamic acid (NMGlu), (c)
Monomethacryloyloxyethyl maleate (MMEM), (d) N-methacryloyl hydroxyproline (NMHyp), and (e)
N-acryloyl aspartic acid (NAAsp). All images were at original magnification of 30003. The images
show that hybridization profiles are similar even though binding expression results do show differ-
ences for the same ligands in Figure 6.

are determined under spatial molecular alignment of func- ture-based model (consisting of topological features such as
tions. The program searches the conformational landscape phenyl ring, carbonyl group etc., chemical functional fea-
of each ligand to identify a large number of its diverse low tures such as hydrogen bond donor/acceptor, acid, base
energy conformations and identifies each ligand as a fea- etc.). The distribution of features in 3D space for a suffi-

Journal of Biomedical Materials Research Part B: Applied Biomaterials

 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN
ciently large number of different conformations (typically
 500) in each ligand is then identified, and the ligand fea-
tures are aligned to best-fit patterns representing common
feature alignment models in subsets of conformations of all
ligands. A specified number of most likely patterns (i.e.,
pharmacaphore models) is thus identified that may realisti-
cally reflect the actual variation in spatial poses of confor-
mations matching complementary 3D alignment of
functions in the target. Each pharmacaphoric pattern is rep-
resented by a center representing the ideal location for each
of the functions and a tolerance sphere of allowed devia-
tions. A vector representing the direction of the bond
between the donor and acceptor locations is also indicated
for hydrogen bonds. An illustrative example of such a phar-
macaphore pattern and the overlay of best-fit conformations
of selected different dentin primer monomers are illustrated
in Figure 8. The results are analyzed by a number of fac-
tors such as the best fit, the number of conformations
among a large set of conformational states (e.g., [250)
with functional placement matching the pharmacaphore pat-
tern and so forth, and other factors which have been
described elsewhere.75
Bonding of Self-Etch Systems to Dentin
In self-etch adhesive systems, etching, and priming steps
are combined into a single step, and in the most recent for-
mulations, etching, priming, and bonding are combined into
a single step. Several investigators have examined the inter-
actions between self-etching systems and dentin. These
adhesives are designed as methacrylated organic acidic
monomers to etch and prime dentin as well as to copolym-
erize with methacrylate adhesive resin. The formulations
are formulated to have an appropriate pH to effectively
condition (or etch) the tissue surface and detach the HAP
phase from dentin matrix. Additionally, the acidic mono-
mer (and its interaction products with HAP, where appro-
priate) may interact with HAP and collagen matrix phases.
The methacrylate functionality also helps polymerize with
the adhesive resin. Therefore both HAP and collagen
phases of dentin may have an important bearing on dentin–
adhesive interactions.
Yoshida et al.,32 examined the interactions of HAP
plates and dentin sections with self-etch adhesives MDP,
PhenylP and 4-META (see Table I) using X-ray photoemis-
sion spectroscopy and transmission electron microscopy
(TEM), respectively. They found that all the self-etch adhe-
sives interacted with the HAP phase yielding similar X-ray
photoemission spectroscopy spectra except for the C 1s
peak, which showed marked differences. On the basis of
the development of C 1s peak, they suggested that MDP
strongly interacted with HAP phase, 4-META less strongly
and PhenylP weakly. Based on electron-lucent interfibrillar
phase observed in TEM, they also suggested that deminer-
alization may only be partial, leaving residual HAP within
the hybrid layer. Similar work by other authors88,89 has
Journal of Biomedical Materials Research Part B: Applied Biomaterials
571
also examined the adhesion mechanism based on adhesive
interaction with HAP and the hydrolytic solubility of the
reaction products. NMR spectra studies by Bayle et al.
have recently indicated that self-etching priming monomers
interact with HAP in dentin through a chemical reaction
leading to the formation of a CaHPO4 structure and a or-
ganic phosphate structure [Ca(RHPO3)2] as follows:90
Ca10 ðPO4 Þ6 ðOHÞ2 þ 8 RPnO3 H2 ! 6½CaHPO4 
þ 4½CaðRHPO3 Þ2  þ 2 H2 O
The CaHPO4 structure may eventually become stabilized as
a brushite crystalline phase (CaHPO4..2H2O). This would
suggest that the electron-lucent phase observed by Yoshida
et al.,31 may include either or both of the brushite and the
HAP crystalline phases. Clearly additional research is needed
to further understand the exact sequence of events.
It is to be noted that collagen priming action will also
be promoted both by the Ca(RHPO3) reaction product and
the original priming agent RPO3H2 in the above reaction.73
Adhesive interaction will therefore be the cumulative effect
of both of these phases. These interactions were computer
modeled by us with respect to typical model structures of
collagen with a [Gly-Pro-Hyp]10 structural sequence modi-
fied with and without a single [Gly-Glu-Lys] or [Ala-Pro-
Hyp] replacement for a [Gly-Pro-Hyp] in the middle repeat
triplet of [Gly-Pro-Hyp]10. AutoDock program was again
used for the analysis. The results have shown that
Ca(RHPO3)2 structure can significantly contribute to the
overall priming interactions with collagen.73
We have also modeled the interactions of HAP with MDP
and PhenylP using AutoDock program. The HAP phase was
a refined model structure modified from the PDB file 35sO2
modeled by Huq et al.,91 Our modeling results indicate that
the interaction energy of PhenylP with HAP phase is signifi-
cantly lower than that of MDP (p \ 0.0001). The binding
energy (kcals/mole) values for MDP ranged from 247.94 to
248.92 in 10 low energy docked conformations although the
range for PhenylP was between 234.55 and 235.52. The
phosphate functionality in all ligands invariably docked right
close to the calcium sites on HAP structure, as seen in Figure
9 confirming strong electrostatic interaction between Ca11
and (PO4)32. The calculated electrostatic energy (kcals/mole)
values ranged between 253.26 and 255.57 for MDP and
238.52 and 240.09 for Phenyl P. This significant difference
in the binding and electrostatic interaction energy values
between MDP and PhenylP is in keeping with the evolution
of C 1s peak detected by Yoshida et al.,32 as indicated ear-
lier. This study is ongoing for other ligands also and will be
reported in a subsequent article.
Limitations of the Theoretical Concepts
Although elegant theoretical advances have been made in
understanding molecular events in dentin bonding phenom-
ena involving adhesive resins, it is important to appreciate
their limitations also. Dentin bonding is a complex problem
572 VAIDYANATHAN AND VAIDYANATHAN

Figure 8. Pharmacaphore hypothesis derived from mapping of preferred 3-D alignment patterns of
low energy ligand conformations. (a) 3-D pharmacaphore map illustrating the best-fit hypothesis of
feature alignment patterns of selected dentin adhesive ligands. Each feature is represented by a
center and tolerance sphere representing potential misalignment range from ideal hypothetical fea-
ture location for the most effective binding. (b) Overlay of ligand lowest energy conformations on
the pharmacaphore model in (a). The ligand molecules in the overlay include 10-methacryloloxy
decamethylene phosphoric acid (MDP), Monomethacryloyloxyethyl maleate (MMEM), N-methacry-
loyl glutamic acid (NMGlu), N-methacryloyl butyric acid (NMBu), N-methacryloyl valeric acid
(NMValer), Bis(2-methacryloyloxyethyl) phosphoric acid (BMEP), 2-methacryloyloxy ethyl phosphate
(MEP), 3-methacryloyloxypropyl phosphate (MPDP), 2-methacryloyloxyphenyl phosphate (PhenylP),
and Dimethacrylglycerol monophosphate (GPDM). The ligands are identified by color code scheme
shown on the left. Although the locations of common functionalities in the selected ligand confor-
mational poses are placed within the pharmacaphore tolerance spheres, misalignments are also
apparent. However, the reasonable fit of the poses to the pharmacaphore hypothesis indicates that
the model is an expression of likely common functional alignment in several ligand binding poses
to the target. [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN 573

Figure 9. Docking of phosphoric acid ligand MDP conformations on HAP crystal. Note that the ligand
conformations dock on the HAP surface with phosphate group (marked by arrow) attached to the cal-
cium sites (pink dots) in HAP. The calculated interaction energy was dominated by electrostatic interac-
tion energy due to the ionic character of the bond and the close distance of approach of the anionic
phosphate functionality of the ligand to the cationic calcium lattice sites on HAP crystal. [Color figure
can be viewed in the online issue, which is available at www.interscience. wiley.com.]

influenced by many factors. Many variables which are not
considered in developing the theoretical concepts may play
a role in bonding events. These include variations in tooth
structure (including differences due to patient differences,
type of tooth restored, location within a single tooth, the
orientation of the fibrils, and the tubules at the cavity prep-
aration segments being restored), operator differences, tech-
nique variations, differences in ambient conditions and
other factors. These differences can potentially significantly
influence the bonding results. Nevertheless, bonding steps
such as etching, priming, and bonding during restorative
procedures and what happens during these steps at the mi-
croscopic and submicroscopic structural levels are also of
major importance for optimum results in efficient bonding.
Durability of Dentin Bonding
Traditionally microleakage has been considered as one of
the major problems associated with poorly bonded restora-
tions.92,93 Bonding to dentin is especially important
because of the relatively permeable structure of dentin in
which a large number of tubular pathways connected to
pulp are embedded in it. With the advances in dentin bond-
ing using an intermediate transitional layer of hybrid and
adhesive resin layers, additional issues such as nanoleakage
and breakdown of the transitional layer between dentin and
the restoration have also become important from the point
of view of clinical durability of restored teeth.
Microleakage
Microleakage is typically related to bacterial invasion
through the restoration-tooth margins and/or pulp causing
short-term and/or long-term clinical problems such as post-
operative sensitivity, marginal staining, secondary caries,
and/or pulpal damage.92,93 To detect possible bacterial pen-
etration, microleakage studies traditionally use tracer mole-
cules, such as dyes, that conform to the size of bacteria
which can percolate only through relatively wide gaps (10–
20 lm in size) which develop between tooth structures and
nonadhesive filling materials, such as composites, amalgam
restorations, cements etc. In composite materials such gaps
often result due to polymerization shrinkage that cause ten-
sile stresses between the cavity wall and the restoration.
The resulting decoupling of the restoration from the cavity
lead to the gaps, and facilitate percolation of bacteria into
the marginal gaps, and from the gaps into the dentinal
tubules and the pulp. A major outcome of the newer dentin
adhesives is the improved sealing of the margins that has
significantly reduced microleakage in restored teeth. This is
because the interconnected structure of primer polymer
chains and collagen fibrils in the hybrid layer and its chem-
ical bonding through copolymerization with adhesive

Journal of Biomedical Materials Research Part B: Applied Biomaterials

574 VAIDYANATHAN AND VAIDYANATHAN
resin layer are effective in closing up the marginal gaps
during bonding procedures. Nevertheless, recent studies by
Waldman et al.,94 and others95–97 have shown that not all
current generation dentin adhesives efficiently seal the mar-
gins. Typically, the adhesive systems described as three-
and two-step total etch and self-etch adhesives exhibit low
microleakage tendency. In contrast, the most recent one-
step all-in-one adhesive systems using self-etching primers
and adhesive resins in a single formulation, exhibit rela-
tively higher tendency for microleakage. A comparison of
the median microleakage scores of a two-step total etch
system [Prime and Bond NT (Dentsply International), des-
ignated as PNB] and a two-step self-etch system [Clearfil
SE Bond (Kuraray, Japan) designated SEB] with that of a
single-step all-in-one adhesive system [One-UP Bond
(Tokuyama Corp., Tokyo, Japan) designated as OUP] was
conducted in our laboratories recently. The microleakage
was evaluated on an interval scale between a low index of
0 (no detectable microleakage) and a highest index of four
(microleakage along the entire margins and through the
tubules into the pulp). The microleakage scores of both
PNB and SEB were centered around a median score of 0,
and that of OUP around the median score close to 2.6. The
OUP system with a low viscosity and high hydrophilicity
adhesive was found to be difficult to retain at all segments
of the cavity wall because of poor thixotropic properties
and exhibited a thick hybrid layer that was devoid of plugs
sealing the tubules and otherwise showing a sharp, rather
than interconnected transition, along the dentin–restoration
interface. Such abrupt phase demarcation at the interface
seems to promote enhanced microleakage.
Nanoleakage
It is well known that primer and adhesive resin may incom-
pletely penetrate the demineralized dentinal collagen layer
after etching in total etching bonding systems.98 This dis-
crepancy between depth of dentin demineralization after
the acid-etching procedure and depth of resin infiltration
facilitates the formation of voids or microporous zones
underneath and within the hybrid layer detectable by silver
nitrate.99–103 As indicated previously, microleakage that
facilitates bacterial percolation occurs only when the gaps
at the margins are 10–20 lm wide. But Sano et al.
observed silver nitrate tracer penetration into the hybrid
layer even when they detected no gaps at the mar-
gins.98,100,101 They also observed that the relatively smaller
tracer ions (silver) penetrated the full thickness of the
hybrid layer in some areas, and into the adhesive resin.
They called this type of leakage, which occurred through
202100 nm gaps at the margins, the phenomenon of
‘‘nanoleakage’’, to distinguish it from microleakage
where leakage occurred through only 10–20 lm wide gaps.
Subsequent studies have shown that self-etch adhesive
systems also manifest nanoleakage within the hybrid
layer. In fact, several investigators have since evaluated
nanoleakage expression in different adhesive systems in
the last decade. Distinctly different types of nanoleakage
patterns (e.g., spotted, reticular, water-treeing) have been
reported by TEM and SEM studies.99,102 Hydrolytic
breakdown of the dentin–restoration bond has been attrib-
uted to the mechanism of nanoleakage expression in the
bonded interface.103
Degradation of Hybridized and Adhesive Layers
The porosities revealed by nanoleakage of tracer ions are
considered as pathways for degradation of the adhesive
interfacial region through water permeation into the net-
work of nanoleakage channels.104–121 The degradation has
also been shown to result in significant decrease in bond
strength over time as well as microstructural evidence of
breakdown.104–120 Degradation may occur by breakdown of
the polymer phase (within the adhesive and the hybrid
layers) or collagen fibrils in the hybrid layer. The resin
phase may be degraded by hydrolysis, which may occur
due to incompletely cured adhesive resin or ester groups in
the polymer chains within the adhesive or hybrid layer.
Salivary enzymes may also theoretically accelerate hydroly-
sis in the resin phase. For example, salivary esterase activ-
ity may accelerate hydrolysis of ester bonds in acrylic
resins. However, the evidence for such accelerated break-
down has been primarily demonstrated in composites.121
No accelerated breakdown of resin-dentin bonds was
observed by esterase activity when it was coupled with col-
lagenase activity and compared against collagenase activity
alone.122 On the other hand, exposure of collagen matrix of
dentin by acid etching may also activate matrix metallopro-
teinase (MMPs),123 which are known to cause collagenoly-
sis in the presence of water. It is known in the literature
that the organization and architecture of collagen fibrils
may determine their tendency to undergo collagenolysis
through MMPs, and typically lone collagen monomers are
more susceptible to proteolytic degradation.124 Conse-
quently, isolated and unprotected collagen fibrils partially
exposed to water in the nanoleakage channels may be espe-
cially vulnerable to collagenolysis through MMPs. When
the collagen fibrils are completely covered by the resin
phase, on the other hand, they are inaccessible to water
needed to effect collagenolysis. According to Carrilho
et al., breakdown occurs on partially exposed collagen
fibrils (not covered by the resin phase) in the hybrid layer
through host-derived MMP-2.125 The activated MMP-2 is
not only known to be an effective gelatinase but also func-
tions as collagenase. Thus, it seems that the long-term
breakdown at the dentin–adhesive interface may undergo
long-term degradation, and there is a need to carry out
additional research to overcome this degradation.
Clinical Performance of Recent
Dentin–Adhesive Systems
Several clinical studies have been reported on recent den-
tin–adhesive systems including total etching, self etching,
and self-etch, self-prime and self-bonding all-in-one sys-
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 MECHANISM OF ADHESIVE RESIN BONDING TO DENTIN
tems.126–137 Most of these studies have clearly demon-
strated that the total etch and self-etch types of adhesives
have provided higher retention rates for restoration in class
V and noncarious cervical lesions relative to earlier adhe-
sive systems. No mechanical retention features were incor-
porated into cavity preparations in any of these studies,
thus demonstrating that the developments in dentin bonding
have advanced the causes of conservative, esthetic, and ad-
hesive dentistry. Postoperative sensitivity is also typically
significantly reduced, especially in the case of self-etching
systems. The clinical evidence is, however, somewhat
mixed for all-in-one formulations with some reports indi-
cating a high success rate in short term studies of some
recent adhesive systems,135–137 whereas other reports indi-
cate retention failure frequencies of 25–49% in clinical
evaluations up to 3 years.129,130 More long-term clinical
studies are clearly needed to assess the durability of recent
all-in-one systems.
SUMMARY AND CONCLUSIONS
Dentin bonding procedures have undergone revolutionary
changes in the last two to three decades. The most signifi-
cant advance has been the recognition that enhanced adhe-
sion between the tissue and the restorative materials at the
interface is critical to ensure durability of the bonds
between the tissue and restorative materials. A transitional
layer that facilitates efficient bonding through an intercon-
nected tissue-adhesive network bonded both to the tissue
and the restoration has been successfully engineered and
developed in the last several years to improve the sealing
and bonding at the tissue-restoration interface. The interac-
tion mechanisms between the tissue and the resin monomer
are complex, but typically involve permeation of the mono-
mer into the demineralized or intact tissue substrate. In the
case of demineralized substrate, Hoy’s solubility parameter
compatibility promotes permeation and secondary bonding
interactions between the substrate and the monomer formu-
lation. In the case of intact dentin, self-etching monomers
demineralize and bond to the mineral phase of HAP. Evi-
dence of potential adhesive interactions between the primer
monomers and the collagen target is also indicated by the
results of computer simulations and immunochemical bind-
ing assays. However, long-term durability of the transi-
tional layer at the interface is partially compromised by the
voids and discontinuities that develop in the transitional
interfacial region across the bonded structure, facilitating
nanoleakage of water into the transitional layer. The inter-
action of water and specific enzymes with the hydrophilic
resins and the collagen fibrils additionally seem to cause
long-term breakdown of the transitional layers. Further
improvements to refine the transitional layer to improve its
adaptation to the intact tissue and enhance its longterm re-
sistance to hydrolysis and breakdown still remain central
challenges for future research. Clinical retention rates of
restorations bonded using the current generation adhesives
Journal of Biomedical Materials Research Part B: Applied Biomaterials
575
have been significantly higher than those of earlier systems
except in one-step formulations where the results are some-
what mixed to date.
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