Biological Aspects of Dental Materials

In studies in adhesive and esthetic dentistry, most research- gest resin monomers, a chronic inflammatory pulp reaction
ers are concerned with the bonding mechanism, adhesion persisted for several weeks after placing the adhesive res-
longevity, and the mechanical properties of the materials. toration. This pattern of pulp response was even more in-
However, for clinical validation, it is very important to know tense when the cavity walls were acid etched before the
the biocompatibility and cytotoxicity of these materials, es- application of bonding agents (Fig 1). The etching of dentin
pecially in relation to the pulp and oral cavity tissues. The with phosphoric acid increased the diameter of the dentin
development of dental adhesives has led clinicians to tubules, dentin permeability, and exudation of dentinal
greatly reduce the use of liners; however, each clinical case fluid, which interfered with the monomer-to-polymer conver- r
deserves special attention and should not be treated with a sion. The low degree of conversion results in a large num-
single protocol. Likewise, whitening techniques using perox- ber of residual monomers that can diffuse through the thin
ide-based agents have become widespread; however, the cavity floor, reaching the pulp at toxic concentrations. It was
risks and limitations of each technique need to be better interesting to observe that patients who underwent the ad-
understood to safely perform esthetic procedures. In this hesive restoration of very deep cavities did not report any
issue of the IAAD Newsletter, Prof. Dr. Carlos Alberto de post-operative sensitivity, which might suggest that such
Souza Costa was invited to comment on these topics. clinical procedure was safe. However, taking into consider- r
ation the persistent chronic inflammation and pulp damage
caused by the application of the adhesive (histological
analysis), our research group has recommended the use of
a biocompatible liner on the floor of very deep cavities. The
data obtained from the in vivo studies also encouraged our
Dental Materials and Pulp research group to investigate the effects of adhesives ap-
Regeneration: Our Contribution plied directly on exposed pulp.
Resin-based products in direct contact with the pulp tis-
I began my academic career as a sue caused a very intense and deep inflammatory reaction
young researcher at the Araraquara mediated by macrophages and giant cells.10-12,17 Several
School of Dentistry – UNESP in 1989, dilated and congested blood vessels scattered on large
where I had the privilege to work in the areas of altered hyaline of the extracellular matrix were
Department of Pathology and surround seen in the coronal and radicular pulp, which exhibited hy- y
myself with supportive and experi- dropic degeneration of cells (Fig 2). Nowadays, many col-
enced faculty. There, I started con- leagues and researchers have reported that all these stud-
ducting in vivo research projects with the aim of assessing ies performed in human teeth, as well as those carried out
the biocompatibility of diverse dental materials. For this in animal teeth and even in pulp cell cultures, were scien-
purpose, I used a few laboratory protocols that were avail- tifically relevant to the fields of operative dentistry and end-
able at the moment. Back then, the financial resources for odontics. The technical developments in these research
research were meager, and I did not have enough scientific areas should not disregard the role and concepts of pulp-
experience to receive grants from the Brazilian Agencies for dentin biology and regeneration.
Research Development for my projects. In collaboration with Since adhesives proved to be unsuitable materials for
other young clinical researchers, in 1995, I began to better application on the floor of very deep cavities or pulp expo-
evaluate the response of human pulps to adhesive restor- r sures, we began assessing other dental materials capable
ations placed in very deep cavities (defined as having a re- of maintaining tissue homeostasis or improving the regen-
maining dentin thickness < 0.5 mm), with or without the eration of damaged pulps. Despite the biocompatibility and
use of a biocompatible liner. The gold standard was a cal- antimicrobial activity of calcium hydroxide (CH) hard-setting
cium hydroxide hard-setting cement. cements, these materials have high solubility, low mechan-
Five years later, we proved that the application of bond- ical resistance, lack of adhesion, and some other deficien-
ing agents directly on the pulp floor disrupted the subjacent cies that encouraged our research group to question
odontoblast layer and killed pulp cells. We also observed whether these products should still be used as liners. Since
the inward diffusion of resin monomers through the dentin then, we have assessed the cytotoxicity of and response of
tubules, which triggered pulp inflammation mediated by human pulps to different formulations of glass-ionomer ce-
mononuclear cells, associated or not with inner dentin ments (GICs). GICs can bond to dentin and enamel, have
resorption in the area related of the cavity floor.4,8,15,33 higher mechanical resistance, better antimicrobial activity,
Based on the fact that such phagocytic cells could not di- and lower solubility than calcium hydroxide cements. While

540 The Journal of Adhesive Dentistry

 a b c

Fig 1 a) Very deep class V cavity prepared in human premolar. The cavity walls were conditioned with phosphoric acid and a bonding agent
was applied. Partial disruption of the odontoblast layer related to the cavity floor is observed. H/E, 125X (CF: cavity floor; RDT: remaining
dentin thickness). b) The blue arrow shows the same blood vessel indicated in Fig 1a. Note the intense chronic inflammatory reaction medi-
ated by monuclear cells and a small area of inner dentin resorption (star). H/E, 250X. c) Stained histologic section obtained from a sound
human premolar which exhibits normal pulp tissue. Observe the tubular dentin and predentin, as well as the odontoblast layer, cell-free zone
and the cell-rich zone. H/E, 250X. This histological image can be compared to the one presented in Fig 1b, in which the pulp-dentin morphology is
changed.

a b c d

Fig 2 a) Six months after capping the pulp exposure with a bonding agent, the human tooth was extracted and processed for microscopic
analysis. Dentin matrix deposition (arrows) is observed around dentin chips (stars) displaced into the pulp during the clinical procedure. How-
ever, the pulp tissue that was exposed to the bonding agent (BA) exhibits a persistent inflammation. H/E, 86X (D: dentin; PE: pulp exposure).
b) Detail of the chronic inflammatory reaction of the pulp tissue capped with a bonding agent. H/E, 125X. c) Center of the coronal pulp of a
human tooth in which the pulpal wound was capped with a bonding agent. Hyaline alteration (HA) of the extracellular matrix among several
dilated and congested blood vessels (arrows) is observed. H/E, 250X. d) Center of the coronal pulp tissue of a sound human tooth. Note the
normal, organized collagen-rich extracellular matrix (EM) among small pulp blood vessels (arrows). H/E, 250X.

resin-modified glass-ionomer cements (RMGICs) cause in- products induces coagulation necrosis, and part of the tissue
tense toxic effects when applied directly to cultured pulp is irreversibly lost. In the process, thousands of pulp cells
cells,7 low toxicity was seen when a thin dentin barrier was die, including resident mesenchymal stem cells (MSCs),
placed between the RMGIC and the pulp cells. Additionally, which play a fundamental role in pulp-dentin regenera-
no pulp damage was observed following the application of tion.5,9,18 Over time, the pulp tissue subjacent to the necrotic
RMGICs on the floor of very deep cavities prepared in layer is capable of healing, but a partially-calcified necrotic
human teeth (Fig 3).6,13,19,23 Based upon these positive area remains interposed between the pulp and the pulp cap-
biological data and taking into consideration the superior ping material. Based on these facts, CH-based products
mechanical/physical properties of RMGICs compared to CH should not be regarded as biocompatible since they cause
cements, our research group recognized RMGICs as the cell death and pulp damage of variable intensity and depth.
best choice of liner cement, irrespective of the thickness of Similar pulp damage also takes place when diverse alkaline
the remaining dentin protecting the pulp. dental materials are used as pulp capping agents, such as
Different formulations of CH have still been widely used Mineral Trioxide Aggregate (MTA) cements.5,9 A few days
as direct pulp capping agents. The highly alkaline pH of such after applying Pro-Root MTA on pulp exposures performed in

Vol 22, No 5, 2020 541

a b Fig 3 a) Very deep class V cavity pre-
pared in a sound human premolar. After
lining the cavity floor with RMGIC, the
lateral cavity walls (CF) were acid etched
and then restored with bonding agent and
increments of composite resin. A very thin
RDT is observed between the cavity floor
and the pulp (P), which does not exhibit
relevant morphological changes. H/E, 86X.
b) High magnification of Fig 3a. The predentin
(arrow) is preserved, the odontoblast layer
is continuous (stars) and no inflammatory
pulp reaction is observed. H/E, 125X.

a b

Fig 4 a/b) Histological section obtained from of a human premolar. Sixty days after capping the pulp exposure with
Pro-Root MTA, the tooth was extracted and processed for histological assessment of the tissue response to the
cement. Components released from MTA reached deep areas of the pulp and concentrated in the local blood vessels
(arrows). H/E, 250X.

human teeth, components of this cement were observed fur- r It also seems important to describe the relevant scien-
ther displaced into this specialized connective tissue.9 With tific contribution that our research group has made in the
time, a Pro-Root MTA component characterized as bismuth last decade to esthetic dentistry, particularly concerning
accumulated in pulp blood vessels (Fig 4). tooth bleaching. Tooth sensitivity has been claimed by most
Our research group has used concepts of tissue engineer- r patients submitted to in-office bleaching treatments, in
ing and molecular biology to develop biocompatible products which gels with high concentrations of hydrogen peroxide
capable of stimulating and driving pulp cells to improve the (30% to 40% H2O2) are applied on enamel for 30–45 min-
pulp-dentin complex regeneration.1,22,24,29,31 After a few utes per session. The intensity of this undesirable side-ef- f
years of research, we developed double-layer scaffolds to be fect, which seems to occur mainly in anterior teeth, declines
used as a direct pulp capping agent (Fig 5a). The inner sur- r with time, and a few days after receiving professional
face of these innovative products, which comes in contact bleaching, the sensitivity is gone. To prevent post-bleaching
with the pulp, exhibits a 3D interconnected-porous structure tooth sensitivity, powerful anti-inflammatory drugs have been
that can be loaded with bioactive molecules and minerals administrated before and after the in-office esthetic treat-
with chemotactic effects on pulp cells (Fig 5b). Pulp cell mi- ment. To determin the origin of post-bleaching tooth sensi-
gration, attachment, proliferation, and differentiation occur tivity and whether this side-effect is related to possible pulp
inside the porous surface of these pulp capping agents, damage caused by the therapy, we preliminarily assessed in
where they synthesize collagen-rich matrix. The outer surface vitro the diffusion of H2O2 from high H2O2-concentration gels
of the scaffolds exhibit dense, impermeable characteristics through enamel and dentin and its effects on pulp cells. The
which prevent the diffusion of toxic components from re- cytotoxic effect of H2O2 was catastrophic.2,3,25,26,28,30
storative materials to the inner bioactive porous surface and Then, we carried out our first in vivo study in which human
the subjacent pulpal wound (Fig 5c). Pulp biology and regen- premolars and mandibular incisors (indicated for extraction
eration is an exciting research field that has driven us and due to severe crowding) were submitted to in-office bleach-
other researchers around the world to develop ground-break- ing.14 The teeth were extracted and processed for histologi-
ing biomaterials with potential to be tested in clinical trials. cal analysis of the pulp response to bleaching. While the

542 The Journal of Adhesive Dentistry

 a b c

Fig 5 a) Lateral view of the sectioned double layer chitosan scaffold (OL: outer
layer; IL: inner layer). b) The OL is characterized as a dense, impermeable barrier
capable of preventing toxic components from reaching the pulp. C) The IL exhib-
its a 3D interconnected-porous structure that can be loaded with bioactive mole-
cules and minerals capable of stimulating the adjacent pulp cells and then
improving the pulp-dentin complex regeneration.

a b
Fig 6 a) Pulp horn of a human premolar
submitted to the in-office bleaching therapy
in which a gel with high concentration of
H2O2 was used. Note that the predentin
(arrows) is preserved and the odontoblast
layer (stars) is continuous. No pulp inflam-
mation is observed. Masson’s trichrome,
86X (P: pulp; D: dentin). b) Pulp necrosis
(PN) is observed in a human mandibular
incisor that was also submitted to in-office
bleaching. The arrows delimit the coronal
pulp area that was occupied by healthy
connective tissue before in-office bleaching.
Masson’s trichrome, 86X.

pulps of premolars did not present any damage or inflamma- H2O2 found in the bleaching gels would produce free radi-
tion, the bleached incisors exhibited large areas of coronal cals and other ROS with higher oxidative potential and a
pulp necrosis (Fig 6). Intense deposition of teritiary dentin shorter half-life than that presented by H2O2 itself. After in-
and inflammation were seen in the radicular pulp. Since teracting with and consequently degrading the intrinsic or- r
then, other studies have been performed with different ganic pigments present in the tooth, these potent new reac-
bleaching agents applied for varying periods of time on tive molecules would be quickly eliminated. Besides
teeth of young and elderly volunteers. In all these clinical obtaining faster in-office tooth bleaching, only a small
studies using human teeth, the histological sections as- amount of residual H2O2 with low potential to cause toxic
sessed under a light microscope showed pulp responses as effects to pulp cells would remain in the tubular dentin sub-
described above, even after only one session of in-office strate. The first in vitro studies confirmed our hypothesis,
tooth bleaching using high-concentration gels.20,21 since the esthetic outcome obtained was appropriate, the
Since then, our research group has worked intensively to transenamel and transdentinal diffusion of H 2O2 de-
develop alternative professional tooth bleaching treatments creased, and the cytotoxic effects caused by the addition of
capable of providing satisfactory esthetic outcomes, such catalytic agents to the bleaching gel decreased statistically
as that obtained by using conventional in-office bleaching significantly.16,27,32 These new data motivated our research
protocols, but without causing tooth sensitivity and pulp group to develop polymeric catalytic primers (PCP) and
damage. We have transferred the knowledge of the Ad- nanofibrillar organic scaffolds (NOS) meant to be applied on
vanced Oxidative Processes (AOP) to esthetic dentistry to the enamel surface before applying the bleaching gels with
develop effective and safe tooth-bleaching treatment. Con- high concentrations of H2O2. The preliminary studies pro-
sidering that H2O2 is a reactive oxygen species (ROS) with vided fascinating scientific data, especially regarding the
moderate oxidative power and a long half-life, we added very low toxicity and the esthetic outcome achieved, even
different enzymes, metallic oxides, and other catalytic when the PCP and NOS were used in association with gels
agents to gels commonly used for in-office tooth bleaching. containing only 10% H2O2. Currently, our research group
We hypothesized that the chemical catalysis of most of has established important collaborations to carry out clin-

Vol 22, No 5, 2020 543

ical trials, which are fundamental to the future use of safer 19. Ribeiro APD, Sacono NT, Soares DG, Bordini EAF, de Souza Costa CA,
Hebling J. human pulp response to conventional and resin-modified
and faster in-office tooth bleaching protocols. The ultimate glass-ionomer cements applied in very deep cavities. Clin Oral Investig
goal is to maintain the excellent esthetic outcomes already 2020;24(5):1739–1748.
20. Roderjan DA, Stanislawczuk R, Hebling J, Costa CA, Reis A, Loguercio
obtained with professional tooth bleaching while avoiding AD. Response of human pulps to different in-office bleaching techniques:
severe pulp damage and consequent tooth sensitivity preliminary findings. Braz Dent J 2015;26(3):242–248.
21. Roderjan DA; Stanislawczuk R, Hebling J, Soares DG, de Souza Costa CA,
caused by the protocols currently used in dental offices. Reis A, Loguercio AD. Histological features of dental pulp tissue from
The topics covered and the scientific data presented in this bleached mandibular incisors. J Mater Science Engineer (A&B) 2014;(4):
178–185.
newsletter, which were obtained in the last three decades of 22. Soares DG, Anovazzi G, Bordini EAF, Zuta UO, Silva Leite MLA, Basso FG,
evaluating the cytotoxic effects and response of human pulps Hebling J, de Souza Costa CA. Biological analysis of simvastatin-releas-
ing chitosan scaffold as a cell-free system for pulp-dentin regeneration. J
to different dental materials, as well as our purpose of devel- Endod 2018;44(6):971–976.e1.
oping innovative biomaterials for clinical use, will be presented 23. Soares DG, Basso FG, Scheffel DL, Giro EM, de Souza Costa CA, Hebling J.
Biocompatibility of a restorative resin-modified glass ionomer cement applied
during the 4th Biennial Meeting of the International Academy in very deep cavities prepared in human teeth. Gen Dent 2016;64(4):33–40.
for Adhesive Dentistry (IAAD). I hope to meet the expectations 24. Soares DG, Bordini EAF, Cassiano FB, Bronze-Uhle ES, Pacheco LE,
of everyone that will attend this high-caliber event. Zabeo G, Hebling J, Lisboa-Filho PN, Bottino MC, de Souza Costa CA.
Characterization of novel calcium hydroxide-mediated highly porous chito-
san-calcium scaffolds for potential application in dentin tissue engineer- r
ing. J Biomed Mater Res B Appl Biomater 2020;108(6):2546–2559.
25. Soares DG, Gonçalves Basso F, Hebling J, de Souza Costa CA. Effect of
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544 The Journal of Adhesive Dentistry

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