d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393

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Biocompatibility of biomaterials – Lessons learned

and considerations for the design of novel
materials

Gottfried Schmalz a,b,∗ , Kerstin M. Galler a

a Department of Conservative Dentistry, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg,
Germany
b Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, Freiburgstrasse 7, CH-3010

Bern, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Objectives. Biocompatibility of dental materials has gained increasing interest during recent
Received 21 December 2016 decades. Meanwhile, legal regulations and standard test procedures are available to eval-
Accepted 31 January 2017 uate biocompatibility. Herein, these developments will be exemplarily outlined and some
considerations for the development of novel materials will be provided.
Methods. Different aspects including test selection, release of substances, barriers, tissue
Keywords: healing, antibacterial substances, nanoparticles and environmental aspects will be cov-
Clinical risk assessment ered. The provided information is mainly based on a review of the relevant literature in
Dentin barrier test international peer reviewed journals, on regulatory documents and on ISO standards.
Pulp healing Results. Today, a structured and systematic approach for demonstrating biocompatibility
Antibacterial subtances from both a scientific and regulatory point of view is based on a clinical risk assessment
in an early stage of material development. This includes the analysis of eluted substances
and relevant barriers like dentin or epithelium. ISO standards 14971, 10993, and 7405 spec-
ify the modes for clinical risk assessment, test selection and test performance. In contact
with breached tissues, materials must not impair the healing process. Antibacterial effects
should be based on timely controllable substances or on repellant surfaces. Nanoparticles
are produced by intraoral grinding irrespective of the content of nanoparticles in the mate-
rial, but apparently at low concentrations. Concerns regarding environmental aspects of
mercury from amalgam can be met by amalgam separating devices. The status for other
materials (e.g. bisphenol-A in resin composites) needs to be evaluated. Finally, the public
interest for biocompatibility issues calls for a suitable strategy of risk communication.
Significance. A wise use of the new tools, especially the clinical risk assessment should aim at
preventing the patients, professionals and the environment from harm but should not block
the development of novel materials. However, biocompatibility must always be weighed
against the beneficial effects of materials in curing/preventing oral diseases.
© 2017 Published by Elsevier Ltd on behalf of The Academy of Dental Materials.

∗
Corresponding author at: Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß-
Allee 11, Regensburg D-93053, Germany. Fax: +49 941 9444981.
E-mail address: gottfried.schmalz@ukr.de (G. Schmalz).
http://dx.doi.org/10.1016/j.dental.2017.01.011
0109-5641/© 2017 Published by Elsevier Ltd on behalf of The Academy of Dental Materials.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393 383

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
2. From single tests to a systematic approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
2.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .384
3. From observation to analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3.1. Release of substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3.2. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .385
3.3. Influence of barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
3.4. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .386
3.5. Influence on cell metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
4. From biocompatibility to tissue healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
4.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .387
5. From cytotoxic antibacterial substances to surface repellants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .387
5.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .388
6. From eluates to nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
6.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .389
7. From classical toxicology to environmental toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
7.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .389
8. From the scientific community to public concern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
8.1. Considerations for novel materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .390
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

1. Introduction
tury [4]. For instance, in 1924 the effect of silicate cement on
Clinical reactions related to dental materials comprise local the dental pulp of dogs had been evaluated [5], furthermore
effects in the direct vicinity, such as inflammation of the the use of amalgam was discussed [4]. In the 60s/70s of last
dental pulp or the adjacent gingiva. Furthermore, gingivi- century the number of such reports increased and different
tis due to increased plaque accumulation e.g. on composite cell culture [6] or animal methods were used for testing differ-
resin fillings has been observed [1]. Allergic reactions to den- ent dental materials [4,7].
tal alloys occur in sensitized patients displaying both extra- However, as outlined above, dental material-related, clini-
and intraoral symptoms. Localized lichenoid reactions of the cally observed adverse reactions are diverse in nature (local,
oral mucosa next to dental materials may also have an aller- allergic, systemic) with different reaction mechanisms behind
gic background. Resins, e.g. from resin based composites and them. Furthermore, dental materials comprise a wide vari-
adhesives may elicit allergic reactions both in dental person- ety of chemically different products including but not limited
nel and in patients. Finally, systemic reactions, where the site to aqueous based cements, resinous materials, mixtures of
of effect is distant from the site of exposure, have been claimed both, metals and ceramics. Additionally, the mode of body
for mercury from amalgam for many years. More recently, contact with dental materials is varying: related to time, it
bisphenol A (BPA) from dental resin composites is subject of ranges from minutes to months and years; related to the site
discussion [2] (see below). Besides these effects, which are of exposure it can be the intact or breached skin/mucosa, it
based on eluted substances or released particles, adverse reac- can penetrate the body surface or it can be placed inside the
tions may be due to physical damage, e.g. tissue burns by light body. Therefore, a more structured and systematic approach
curing units [3]. Thus, biocompatibility of dental materials and was deemed necessary. In the 70s and 80s of last century a set
devices is a clinically relevant topic and has raised increased of tests for demonstrating biocompatibility was issued by the
interest in the dental scientific community, the profession and American Dental Association (ADA) as Specification No. 41 and
in the public. Herein, the experiences and developments from by the Fédération Dentaire Internationale (FDI) as Technical
the last four decades of biocompatibility evaluation of dental Report No. 9 [4,8]. Later, different and more specified stan-
materials will be exemplarily delineated and conclusions will dards were developed (see below). Legal regulations defining
be provided with respect to the design of new materials. dental materials as medical devices and implementing safety
requirements were initiated 1976 in the US, followed by the EU
in 1993 and are existent virtually worldwide today [4]. In these
2. From single tests to a systematic legal regulations, the safety requirements are based on risk
approach classes, usually ranging from I to III, where class I denominates
low risk and class III high risk [1,9].
Tests on material-related adverse biological reactions and Eventually, it became clear that a set of tests with differ-
attempts to prevent them could be found only sporadically ent endpoints needed to be performed in order to provide full
in the literature from the beginning to the middle of last cen- information on the “biocompatibility” of a material [1]. For
384 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
Fig. 1 – Compositions of the surface and the bulk of a dental alloy are significantly different. The release of elements reflects
the composition of the surface, but not of the bulk [16].
dental materials the following endpoints are part of most of
the test programs:
a Cytotoxicity (in vitro).
b Genotoxicity/Mutagenicity (mainly in vitro).
c Local reactions (experimental animals or in vitro simulation
tests like the dentin barrier test, see below).
d Sensitization (mainly experimental animals).
Other endpoints such as systemic acute, subchronic or
chronic toxicity, cancerogenicity or reproductive toxicity have
to be considered [9]. Some of these tests require a high num-
ber of, or large, test animals. According to the concept of
Threshold of Toxicological Concern (TTC) such endpoints may
also be evaluated based on the amount of eluted substances
from a material [10,11]. For cytotoxicity evaluation in this con-
text, usually permanent cell lines like 3T3 or L-929 fibroblast
are used together with unspecific cell viability endpoints (e.g.
MTT, MTS or neutral red assays).
2.1. Considerations for novel materials
It is obvious that a systematic approach for the evaluation of
the biocompatibility of novel materials/substances is neces-
sary and that toxicological knowhow must be included already
at an early stage of material development. The final choice on
the tests that have to be performed is based on a so-called
Clinical Risk Assessment as delineated in ISO 14971 [12] and
ISO 10993-1 [9].
The ISO 10993 series has been developed for all medical
devices (including dental materials) and rules for the selection
of the appropriate test methods as well as the methods them-
selves are described. By now, this Standard comprises 20 parts
and additionally a number of informative Technical Specifica-
tions/Reports. ISO 7405 [13] is designed especially for medical
devices used in dentistry and should be applied together with
the ISO 10993-1 [9]. In conclusion, ISO standards play an impor-
tant role today, both in risk assessment and implementation
of testing as well as in the evaluation of test results, because
they can be used to meet the legal requirements for premar-
ket biocompatibility evaluation. Therefore, wherever feasible,
standard tests should be applied in an early stage of material
development, where the new material should best be tested
against a market product (relative toxicity analysis). As all pre-
clinical tests used for certification/market access have certain
limitations, biocompatibity evaluation must finally be com-
plemented by a post market information systems that require
dentists to report adverse effects in their patients either to the
manufacturer or to other bodies and which are an important
part of the whole risk management system [12].
3. From observation to analysis
3.1. Release of substances
As mentioned above, biocompatibility testing of dental mate-
rials in the past was focused on a description of observed
effects on live tissues, such as cell death or inflammation.
However, in the course of developing a more structured
approach to biocompatibility evaluation and in order to
improve material biocompatibility, an analysis of the causes
of such reactions became necessary. It was generally accepted
that the basis for biocompatibility evaluation is the analysis
of released substances from a dental material (eluate). Both
the total amount of released substances and the composi-
tion of the eluate should be evaluated [1,9,14]. This release
is, however, not directly related to the composition of the
material. For example, for many resin composite materials the
organic phase is largely composed of mixtures of bis-GMA and
TEGDMA. Although the total amount of bis-GMA is higher,
the more hydrophilic TEGDMA is generally eluted in greater
amounts in aqueous solvents [15]. Furthermore, the composi-
tion of a material may be different in the bulk compared to the
surface. An example for an alloy is shown in Fig. 1: the surface
and bulk of the alloy reveal a significantly different composi-
tion. The surface layer contains relatively small amounts of
gold, but a high percentage of copper and silver. This correla-
tion is reflected by the elements that are released (primarily
copper) after 24 h [16]. Another example is that – after firing
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
– alloys used for metal ceramic crowns change their surface
composition, but not the bulk composition. The release of
metal ions increased significantly, as does cytotoxicity [17].
Therefore, actual data on the release of substances are nec-
essary as a basis for the evaluation of biocompatibility of each
material.
It remains a matter of discussion whether the amount of
eluted substances from polymerized resin materials is cor-
related with the conversion rate. According to Ferracane, a
weak correlation exists between conversion rates and eluted
residual monomers, if water is used as extraction medium.
A stronger correlation was found for a mixture of water with
ethanol [18]. Tanaka et al. [19] asserted that a clear correlation
exists between conversion rate and the release of TEGDMA
and Bis-GMA into an aqueous medium. However, for bifunc-
tional acrylates the inclusion of only one functionality into
the network is sufficient to prevent the molecules from elu-
tion. Therefore, the terms “degree of conversion” and “eluted
substances” should be used separately.
In this context it needs to be mentioned that the amount
of eluted substances is also dependent upon the preparation
of the test samples. Thus, surface morphology of test sam-
ples should be standardized, because the release of substances
also depends on the amount of available surface. For resin
based materials, the oxygen inhibition layer on the surface
will influence the amount and composition of the eluate [20].
3.2. Considerations for novel materials
The basis for any evaluation of biocompatibility is the deter-
mination of substances liberated from a new material. In this
case as well, relevant standards can be applied such as ISO
10993 parts 12 and 18 [14,21]. The relation of sample surface
to volume of solvent should comply with ISO 10993-12 [21] and
at least a hydrophilic (e.g. saline) and a lipophilic solvent like
ethanol or methanol should be used. Additionally, mixtures of
75% ethanol and 25% water have been proposed [1,22].
Relevant methods of chemical analysis of materials and
eluates are described in ISO 10993-18 [14]. Details for sam-
ple preparation for dental materials are described in ISO 7405
[13] and should be meticulously followed and reported. How-
ever, the users need to realize that although the release of
substances from a biomaterial is the prerequisite for a bio-
logical reaction, this does not mean that the mere presence
of eluted substances in the tissues makes the material non-
biocompatible. Other factors, especially the concentrations
are also important. Nevertheless, the reduction of eluted sub-
stances from a new material should be considered during the
developmental process.
3.3. Influence of barriers
If a residual dentin layer of more than 0.5–1 mm was present,
no apparent pulp damage was observed in pulp studies with
resin composite materials both in experimental animals and
in humans [1], although dentinal adhesives or some resin
composites are cytotoxic in direct contact with cells [23]. The
same is true for zinc oxide and eugenol: it is strongly cytotoxic
after direct contact with cells [24], but with a residual dentin
layer of >0.5 mm no apparent pulp damage can be observed
385
Fig. 2 – Dentin permeability (measured as hydraulic
conductance) for different dentin thicknesses [1].
histologically [7], which justifies its use as a non-toxic refer-
ence material in ISO 7405 pulp studies [13].
In a number of studies it was shown that dentin has a pro-
tective effect. Dentin acts as a diffusion barrier for substances
liberated from dental materials [25]. Permeability of dentin has
been proven to be low, but it increases exponentially with a
residual dentin thickness of less than 0.5 mm [26] (Fig. 2). Espe-
cially lipophilic substances like eugenol liberated from a zinc
oxide and eugenol mix diffuse at a very limited rate through
the hydrophilic dentin; thus the concentration at the cavity
floor is high enough to kill bacteria, but low enough in the pulp
where no damage can be observed after histological analysis
[1,27].
Furthermore, dentin was found to be able to bind eluted
substances from restorative materials, e.g. eugenol, which
is bound to dentin components like calcium apatite and
to certain proteins, such as albumin [28]. Discoloration of
the dentin floor under unlined amalgam fillings shows that
metal ions from amalgam become bound to dentin. Further-
more, dentin is able to consume protons when in contact
with acids. Therefore, a time limited (30 s) application of
(citric) acid on dentin of >0.5 mm thickness did not signif-
icantly increase dentin permeability [26] (Fig. 2), because
after a limited surface demineralization the available protons
were consumed and no deeper demineralization occurred.
Interestingly, we found that the antibacterial effect of a self-
etch dentin primer (here against Streptococcus mutans) was
blocked after contact with dentin (see Fig. 3), which may
be attributed to the fact that antibacterial protons were
consumed by dentin and then were no longer available
for inhibiting/killing bacteria. Dentin also acts as a bar-
rier against heat, which is produced e.g. by the exothermic
reaction of resin composites [29] and additionally by light cur-
ing units [30–32]. Thermal transfer increased exponentially
with decreasing dentin thickness [30].
On the other hand, dentin contains odontoblast processes
in the dentinal tubules. During cavity preparation these cel-
386 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393

lular processes become injured. However, with a thickness of

the dentin layer of >0.5 mm this cell injury apparently does
not lead to further pulp damage beside the formation of some
reactionary dentin in the pulp at the end of the severed tubules
[33]. Furthermore, dentin under a carious lesion undergoes
sclerosis and thus it can be assumed that the number of sev-
ered odontoblast processes is reduced.
Interestingly, dentin does not seem to be an effective bar-
rier against bacteria and bacterial products that are present
between resin composite filling and the bottom of the cavity
[34]. Such bacteria have consistently been found in cases of
pulp inflammation under resin composite fillings, also if the
residual dentin layer was larger than 1 mm [1]. This means Fig. 3 – Antibacterial activity of an acidic dentinal adhesive
that a tight seal of a cavity by an adhesive restoration is con- primer without (Clearfil SE Bond) and with (Clearfil Protect
sidered to be protective against pulp damage. Technical and Bond) DMPB against S. mutans ATCC 25175 tested in an
biological effects are interrelated. agar diffusion test; the selfetch primer without MDBP loses
The oral mucosa is composed of keratinized or non- its antibacterial effect in contact with dentin (for details of
keratinized epithelial cells, a base membrane and fibroblasts the test method see [63]).
and it acts as a barrier concerning the resorption e.g. of
metal elements or nanoparticles. Several methods have been
described to simulate this situation in vitro. A three dimen-
sional culture of epithelial cells from an oral carcinoma grown
on a polycarbonate filter was used as a reconstituted human
epithelium model [35], a different study used co-cultures of
human fibroblasts and keratinocytes [36]. Other methods are
based on immortalized epithelial cells and fibroblasts grown
on a mesh [37]. An animal test method using the hamster
pouch has been described [38] and included into the Annex
Fig. 4 – The dentin barrier test according to ISO 7405 is
of ISO 10993-10 [39]. However, experience in the published lit-
based on a split perfusion chamber (left), in which the test
erature with all these methods for dental materials is rather
material and the cell culture is separated by a dentin disk
limited.
[13,40].

3.4. Considerations for novel materials

If a novel material is cytotoxic, this does not automatically
mean that it is not suitable as a filling material. It must
be analyzed further whether the toxic substances have the
potential to diffuse through dentin in sufficient concentra-
tions. In such cases, the indications should be restricted, e.g.
with the exclusion of direct pup capping. This also means that
if the biological properties of novel materials are tested, barri-
ers between materials and target cells should be implemented;
in case of filling materials this would be dentin.
In order to mimic this situation, we developed the dentin
barrier test [40,41], which can be used as an in vitro simula-
tion test for cytotoxicity evaluation of novel dental materials
intended to be used for cavity filling. In this test, a split perfu-
sion chamber is divided into two compartments by a defined
dentin slice. On one side of the dentin slice the test material
is applied, on the other side a three-dimensional cell cul-
ture is inserted (Fig. 4). With this method, zinc oxide eugenol
showed no cytotoxicity, which is in agreement with clinical
experience [40]. On the other side, a RMGIC elicited a toxic
reaction [40], which shows that the system reacts. This RMGIC
was found to damage the pulp tissue when applied in deep
cavities of <0.3 mm residual dentin thicknesses [42]. Further-
more, the protective effect of increasing dentin thickness was
demonstrated for a RMGIC in this test system [43]. This test
method has meanwhile been included into the ISO 7405
(Annex) [13]. Similar test methods need to be evaluated for
measuring increases in temperature e.g. due to the setting
reaction of resin composite materials and the additional effect
of light curing units. Some in vitro test have been described
[32], but information on the correlation to in vivo studies
seems insufficient [44].
Concerning the epithelial barrier, the passage of molecules
and nanoparticles (see below) through such barriers are a topic
of interest as these particles from materials but also from the
ingestion of tooth pastes get into contact with the oral mucosa.
Interestingly, some dental alloys have been used successfully
in contact with the oral epithelium, but they were not biocom-
patible when used as implants [16].
3.5. Influence on cell metabolism
As a further step toward a better understanding of material
tissue interactions, the influence of substances from dental
materials on cellular metabolism was investigated at concen-
trations of the toxicant that are low enough to not lead to cell
death or gross growth inhibition. Apparently, at concentra-
tions of acrylate monomers (in this case HEMA and TEGDMA)
which do not impair gross cell vitality and cell growth, cellular
glutathione is consumed in order to detoxify the monomers
[45]. After consumption, glutathione is no longer available
to balance the production of reactive oxygen species (ROS)
from normal cell metabolism, which results in a ROS imbal-
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
Fig. 5 – Alkaline phosphatase activity (Y1-axis) and cell
numbers (Y2-axis) of corresponding samples for cells and
cells with and without 0.3 mM TEGDMA for 14 days
(medians with 25th and 75th percentiles). ALP activity is
normalized to cell numbers in corresponding samples and
expressed in nmol/1000 cells/1 h. Whereas cell numbers in
treated samples and controls were nearly identical, ALP
activity was significantly reduced by TEGDMA [53].
ance [45–47]. The increase of ROS leads to the oxidation of
DNA (generation of 8-oxoguanine) and to genotoxic effects
[48]. Furthermore, the cell cycle is delayed/arrested and even-
tually, apoptosis is induced, if the DNA damage is beyond
repair [46,49]. Due to the increase of ROS, cellular enzyme sys-
tems are differentially up- or down regulated (adaptive cell
response) in order to compensate for the imbalance of ROS
[46,50]. Such processes consume energy and may lead to early
cell senescence. The clinical implications are described below.
4. From biocompatibility to tissue healing
Initially, a biomaterial applied on a breached surface or a
surgical wound should, first of all, not damage the tissues (fur-
ther). However, this concept has been extended with the aim
that the materials should also not interfere with the healing
process. Apparently, the borderline between biocompatibil-
ity, impairment of healing and bioactivity are fluxionary. For
the exposed pulp – in analogy to skin wounds – healing may
imply the formation of a new barrier, in this case dentin.
However, in direct contact with adhesives/resin composites,
the dental pulp does not produce new dentin, which can
be considered as an impairment of the intended healing
process [1,51]. Interestingly, the level of accompanying pulp
inflammation in such cases has partially been reported to
be rather low [52]. In cultures of pulp derived cells exposed
to a monomer at concentrations that did not impair cell
growth the normally occurring biomineralization (indicated
by alkaline phosphatase activity in controls without monomer
exposure) was not observed [53] (Fig. 5). As mentioned above,
at monomer concentrations, which do not kill cells or impair
gross growth, these cells are subject to stress and senescence,
which may be the reason that the normally occurring process
387
of reparative dentin formation including stem cell prolifera-
tion, migration and differentiation is impaired.
Hydraulic calcium silicate cements (HCSCs) on the other
side support pulp healing processes. Originally, materials
derived from Portland cement had been used. Recently, simi-
lar materials have been introduced to the market, which are
based on chemical components such as tricalcium silicate. In
vitro, these materials were less toxic to a three dimensional
culture of pulp-derived cells than a glass ionomer cement and
were able to upregulate marker genes of biomineralization
[54]. This data is in line with results from other in vitro [55,56]
and in vivo studies [57].
4.1. Considerations for novel materials
HCSCs have the potential to induce pulp healing. Furthermore,
the use of antioxidants like N-acetyl cysteine e.g. together
with acrylate monomers should be considered [58]. For novel
materials intended to be used on breached surfaces or with-
out any barrier, like on the exposed pulp, specific endpoints
for analysis are necessary and therefore tests described in
the above mentioned standards are not the prime choice.
For instance, for the exposed pulp, biomineralization mark-
ers such as alkaline phosphatase, dentin sialophosphoprotein
(DSPP), or dentin sialoprotein (DSP) are relevant. For cell cul-
ture experiments specific target cells, like pulp derived cells
should be used.
5. From cytotoxic antibacterial substances
to surface repellants
Antibacterial activity is generally regarded as a desirable
property of dental materials. As has been mentioned above,
bacteria under resin composite restorations have been held
responsible for pulp damage [34]. Furthermore, increased
plaque accumulation on resin composites restorations can be
associated with the onset of gingivitis [1]. Also, for resin based
denture materials bacterial adhesion is a matter of concern
[59].
Bacterial adhesion/accumulation on and under materials
can be reduced or prevented by different measures, e.g. polish-
ing or regular and effective oral hygiene. Another commonly
used method is to introduce antibacterial substances into
materials. These exert their effects mainly after elution of
the active substance. In the past, a large number of sub-
stances has been incorporated into dental materials in order
to achieve these properties, e.g. copper, zinc, silver, fluorides,
cetylpyridiniumchlorid, different chlorhexidine compounds,
glutaraldehyde, triclosan, zinc oxide and eugenol, and antibi-
otics [58]. Addition of microparticulate silver in commercially
available dental adhesives may have the potential to reduce
secondary caries [60]. However, several problems are afflicted
with this approach. The elution may have a negative effect on
the material’s physical/mechanical properties [61]. Finally, due
to their unspecific mode of action, antibacterial substances
are also cytotoxic and, therefore, biocompatibility becomes a
matter of concern.
A solution for preventing or reducing the negative effect of
the inherent cytotoxicity of antibacterial substances clinically
388 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
Fig. 6 – Chemical structure of SAPYR containing a positively
charged pyridinium-methyl substituent [64].
is the time limitation of action: for a short time the substance
is actively displaying its antibacterial and cytotoxic effects,
thus eliminating or reducing the bacterial load; however, in
the long term the material is biocompatible.
An example is the antibacterial molecule methacryloyloxy-
dodecylpyridinium bromide (MDPB), which was introduced by
Imazato et al. [62] to be incorporated e.g. into a dentinal adhe-
sive. The antibacterial adhesive proved to be more effective in
dentin than chlorhexidine [63]. However, this activity stopped
after light curing preventing pulp damage.
Another approach for time limited effect is the photody-
namic inactivation of bacteria. With this method, a special
photosensitizer is needed, which is activated by irradiation
with light of a defined wave length. Without light activation,
the photosensitizer is non-toxic. Current photosensitizers
used in dentistry are mainly colored (e.g. methylene blue)
and special light sources are needed [64]. Recently, we have
developed a new colorless photosensitizer (Fig. 6), which can
be activated with a light curing unit for composite resins.
Used together with a standard light curing unit (Bluephase C8,
Ivoclar Vivadent) this substance proved to reduce the num-
ber of bacteria (Enterococcus faecalis, Actinomyces naeslundii and
Fusobacterium nucleatum) on mono- and polyspecies biofilms
by about 3–5 log levels [64]. Interestingly, for bacteria with
an endogenous photosensitizer like porphyrin, the irradiation
with light alone (470 nm, 150 J/cm2 ) had an antibacterial effect
[65].
Another promising method for preventing the colonization
of bacteria on or under a material surface is to modify the
surface chemistry. As mentioned above, polymerized MDPB
exerted no antibacterial effect [63]. However, the positively
charged pyridinium group on the surface has the potential to
be biologically active. When the MDPB molecule was chemi-
cally bound to a silicon wafer surface, the adhesion of bacteria
was not inhibited, but most of the bacteria were dead. Unfortu-
nately, this effect diminished after the MDPB modified surface
was incubated with saliva, serum or albumin [66]. In a fol-
low up we used an amplification of surface functionalities per
unit surface by immobilizing polyamidoamine (PAMAM) den-
drimer molecules on wafer surfaces. The bacteria-repellent
behaviour of immobilized PAMAM-NH2 was maintained even
after conditioning with saliva [67].
The use of different surface morphologies by incorpo-
ration of microparticulate substances to prevent bacterial
adhesion is presently under investigation [68,69]. The influ-
ence of hydrophobic/hydrophilic surface properties (contact
angle, wettability) on bacterial adhesion is a matter of dis-
cussion. Saliva coating which produces a pellicle influenced
both wettability and bacterial adhesion; it mainly decreased
the wettability of dental materials. Whereas without prior
saliva coating of the test samples a linear relation was found
between increasing contact angle (water) and bacterial adhe-
sion (Streptococcus gordonii DL1), this was no longer apparent
after preincubation of the material samples with saliva [70].
Apparently, the composition of the pellicle that provides spe-
cific ligands for bacterial adhesion may play a major role [70].
5.1. Considerations for novel materials
The examples mentioned here shall demonstrate that inte-
grating antibacterial properties into novel dental materials is
still a major challenge also with respect to biocompatibility.
Elution based approached have been frequently attempted,
but mainly with limited success. New approaches have been
proposed, but to date, data are insufficient to allow for clin-
ical applications. In any case, if testing antibacterial effects
of novel materials, the influence of saliva has to be taken
into account. Finally, antibacterial effects relying on surface
effects rather than on eluted substances may have a better
perspective concerning their biocompatibility.
6. From eluates to nanoparticles
In addition to the concept that biological reactions are based
on a chemical interaction of an eluted substance with biologi-
cally relevant molecules, the role of engineered nanoparticles
has more recently become a matter of interest. Such parti-
cles have a size (at least in one dimension) of 1 nm–100 nm
[71]. According to a definition of the European Commission
[71] the term nanomaterial means a natural, incidental or
manufactured material containing particles, in an unbound
state or as an aggregate or agglomerate in which, for 50% or
more of the particles in the number size distribution, one or
more external dimensions is in the size range 1 nm–100 nm.
The biological effects of nanoparticles and nanomaterials in
general cannot be outlined here and the reader is referred to
relevant reviews; e.g. [72,73]. A main problem of toxicological
concern is the fact that these small particles can penetrate
the membranes of cells and cellular organelles. The Scientific
Committee on Emerging and Newly Identified Health Risks
(SCENIHR) of the EU Commission has recently issued a report
on Guidance on the Determination of Potential Health Effects
of Nanomaterials Used in Medical Devices [74].
During any industrial grinding process, nanoparticles are
produced as by-products, which are small in weight but large
in relative number. As the EU-definition of a nanomaterial (see
above) is based on the relative number of nanoparticles, many
dental materials (e.g. containing any filler, pigments, etc.)
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
are nanomaterials. However, such nanoparticles are bound
in the matrix and not intended to be released. Furthermore,
nanoparticles are incorporated into surfaces of some materi-
als, e.g. of dental implants. Only few materials contain fine
and ultrafine particles (<0. 1 ␮m), which are intended to be
released, for instance scanning sprays [75]. Nanoparticles are
also included in many other products of our daily life, like sun
blockers or toothpastes.
Nanoparticles may be released e.g. during intraoral grind-
ing of resin composite materials [76] and in the dental
laboratory. Interestingly, such particles may also be produced,
although the material itself does not contain nanoparti-
cles [77]. Furthermore, nanoparticles in cosmetic products
like tooth pastes are swallowed and particles from titanium
plate surfaces have been detected in neighboring tissues
[78]. In experimental animals, titanium particles in regional
lymph nodes after insertion of titanium screw implants
have been found [79]. A risk assessment for the differ-
ent exposure routes (e.g. inhalation, swallowing, via oral
mucosa or direct tissue contact) is necessary, where inhala-
tion is presently regarded to be the most relevant. Generally
accepted limit values for nanoparticle exposure do not exist.
Alternatively, fine dust exposure limit values for occupa-
tional exposure (e.g. 1.25 mg/m3 for dust reaching the alveoli,
Germany, TRGS 900-2014) can be used, although they refer
to much larger particle sizes in the micrometer area. In a
more recent German recommendation, a limit for (theoret-
ically) pure nanoscale dust was calculated to be between
110 and 190 ␮g/m3 (www.baua.de/ags, Beurteilungsmaßstab
NanoGBS). However, these data are mass based and they do
not take into consideration that the relevant dose might be
the particle number and not the mass. In a literature review,
Terzano et al. reported that on a so-called “low pollution” day
(over 24 h), an adult human will inhale approximately 200 bil-
lion particles (about 400 ␮g), half of which will be deposited in
the lung, without apparent harm [73]. In this context, van Lan-
duyt et al. [76] found short episodes of 105 –106 particles/cm3
being produced when reshaping and contouring three front
teeth composite veneers with rough polishing disks and dia-
mond burs (background exposure 5000–10,000 particles/cm3 ).
The actual risk for patients and the dental personnel (risk
group) needs further to be evaluated and wet grinding has
been recommended whenever possible.
6.1. Considerations for novel materials
Nanomaterials have significantly improved material proper-
ties. So far, there is no reason to not use nanoparticle e.g. as
fillers in dental materials. The new draft of the Medical Device
Regulation (MDR) of the European Union [80] does not gen-
erally regard nanomaterials as Class III devices (as initially
intended), but the assignment of a material to the risk classes
is based on the potential for internal exposure to nanoparticles
of each single product. Within the clinical risk assessment,
the risk deriving from nanoparticles must be addressed. Model
calculations should be performed for a rough estimate of the
exposure (dental personnel and patient) by intraoral grinding
or wear (patient) for dental materials.
389
7. From classical toxicology to
environmental toxicology
During recent decades, environmental aspects of man-made
products have attracted considerable attention. In this con-
text, mercury in general but also from dental amalgam had
been a matter of discussion. In 2013 the Minamata Conven-
tion [81] was adopted with the aim to generally reduce the
discharge of mercury into the environment. This also included
the use of amalgam, for which a phase down strategy was
adopted with no time limit.
From amalgam waste, mercury can reach the environment
and through the food chain indirect effects on humans were
considered possible. The Scientific Committee on Health and
Environmental Risks (SCHER) of the European Commission
issued a report on the environmental risks and indirect health
effects of mercury from dental amalgam in 2015 [82]. SCHER
concluded that, under extreme local conditions (maximal
dentist density, maximal mercury use, absence of separator
devices), a risk of secondary poisoning due to methylation
cannot be excluded. However, this can be compensated by
the use of effective separators. Also in the Minamata conven-
tion, the use of separator devices is listed as one provision
for the amalgam phase down. Regarding the risk for human
health due to environmental mercury in soil and air origi-
nating from dental amalgam use, SCHER concluded that this
emission fraction of Hg represents a very minor contribution
to total human exposure from soil and through inhala-
tion. On the other side, information available on the Hg-free
alternatives does not allow a sound risk assessment to be per-
formed [82].
Here, bisphenol A (BPA) has attracted considerable concern.
BPA is an estrogen mimic binding to the estrogen receptors ER␣
and ER␤. It has been claimed that BPA leads to reduced fertil-
ity, irreversible changes during child development (influencing
time of puberty), is neurotoxic, or induces diabetes and adi-
positas [83,84]. Recently it was shown in experimental animals
that BPA administered at a specific stage of pregnancy caused
Molar-Incisivi Hypoplasia and may exacerbate the appear-
ance of dental fluorosis [85–87]. The clinical relevance of these
studies on experimental animals is not clear yet and more
research is necessary. Although BPA is not used as such in
dental materials, it is used during the production process of
some commonly used monomers, such as Bis-GMA. There-
fore, impurities are present and are eluted [88]. However, the
amount of BPA eluted was orders of magnitude lower than the
limit values recently proposed by the SCENIHR report of the
European Commission [88,89]. SCENIRH evaluated the role of
BPA in medical devices and concluded that the long-term oral
exposure via dental materials is far below the recently derived
temporary oral external TDI of 5 ␮g/kg b.w./day derived from
animal studies and poses no risk for human health. In addi-
tion, short-term (relatively high) exposures to dental materials
are below this recently established temporary TDI (t-TDI) [90].
7.1. Considerations for novel materials
The extension of the term biocompatibility to include envi-
ronmental aspect will not be limited to amalgam. It is to be
390 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393
Fig. 7 – Overview of the present concept of material tissue interaction.
expected that the possible impact of bisphenol A and other
estrogen mimicking substances will become a topic of con-
cern. The possibility of developing materials devoid of possible
endocrine disruptors should be considered.
8. From the scientific community to public
concern
In contrast to most other dental material properties, discus-
sions on the biocompatibility are not limited to the scientific
and professional community, but this topic has gained high
public interest and concern. Through the internet lots of –
uncontrolled – information on actual or only claimed adverse
effects of dental materials reach a broad public. This was espe-
cially evident with the discussion on amalgam. Although the
scientific data – as evidenced by many national and interna-
tional scientific reports – show that for the general population
amalgam is a safe material (e.g. SCENIHR [91]), the pub-
lic has a strong distrust. The arguments put forward in the
public discussion are similar for amalgam and resin compos-
ite materials partially based on not substantiated claims. In
some countries like Norway the use of amalgam is virtually
totally restricted due to environmental concerns. However,
environmental concerns have always to be weighed against
the consequences of restricting the use of materials on health
effects. Here, the situation in different parts of the world has
to be taken into account, as delineated in the Minamata con-
vention [81].
8.1. Considerations for novel materials
The considerable public concern calls for a common strategy
of risk communication for the manufacturer and the dental
community. This should include not only one material, but
also possible alternatives. It should be kept in mind that den-
tal materials are used for treating a disease. On the other
side, the concerns of the patients should be taken serious.
The dentist should, however, have the relevant information
at hand to be prepared for such a discussion (argumentative
competence). The dentist should be the competent contact
person, who responds to the information available (e.g. from
the internet) and transfers it to the clinical context of the
specific patient situation. Sufficient information regarding the
material composition of dental materials applied in the oral
cavity of the patient are an important prerequisite. Finally it
should be stressed that biocompatibility of a materials must
always be weighed against its benefits in curing/preventing
oral diseases.
9. Conclusions
Biocompatibility of dental materials has become a complex
issue (Fig. 7) and a matter of concern for patients, profes-
sionals, regulatory authorities and the public. Thus it has
become an important aspect for the development of new
materials and substantial additional resources are needed. A
large number of legal regulations are effective and relevant
standards available. Biocompatibility considerations must be
included at an early stage of new material development. The
special application of a material must be taken into account
when defining the necessary tests, evaluating the results and
determine the indications for use. Post market information
systems are an important part of securing final biocompatibil-
ity. As important as new materials are new biocompatibility
test methods with better predictability [92]. The benefits of
increased safety for patients, professionals and the public are
obviously difficult to quantify. However, increased knowledge
on the biocompatibility of dental materials has certainly led
to a better understating of material tissue interaction, to the
development of new materials and practically relevant safety
precaution measures. A wise use of the new tools, especially
the clinical risk assessment aims at preventing the patients,
the professionals and the environment from harm on one side
but not blocking the development of novel materials on the
other.
 d e n t a l m a t e r i a l s 3 3 ( 2 0 1 7 ) 382–393 391

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