DOI: https://doi.org/10.54617/adoklinikbilimler.

1401043

Review
Evaluation of Biocompatibility Properties of Dental
Materials: xCELLigence® System
Dental Materyallerin Biyouyumluluk Özelliklerinin
Değerlendirilmesi: xCELLigence® Sistemi

Makbule Buse Dündar Sarı

ABSTRACT ÖZET
The toxic and biological impacts of dental materials play a pivotal Diş hekimliğinde dental materyallerin toksik ve biyolojik etkileri kli-
role in their clinical application within dentistry. The assessment of nik kullanımda büyük bir öneme sahiptir. Dental materyaller yeni
these materials typically commences with in vitro tests upon initial geliştirildiğinde canlı dokulardaki etkisi, etik ve yasal yükümlülük-
development, progressing to in vivo animal experiments and ler nedeniyle öncelikle in vitro testler sonrasında in vivo hayvan
clinical trials. In vitro cell culture tests afford the examination of deneyleri ve klinik deneyler ile değerlendirilmektedir. İn vitro hüc-
tissue responses at the cellular level, allowing the observation of re kültürü testleri ile dokuların hücre düzeyinde yanıtları incele-
physiological activities. Moreover, these tests offer a cost-effective nebilmekte ve fizyolojik aktiviteleri taklit edilebilmektedir. Ayrıca
and time-efficient alternative to animal experiments, rendering hücre kültürü testlerinin hayvan deneylerine göre maliyeti daha
them easily applicable and replicable. Recently, real-time cell düşüktür. Daha kısa süre almakta, kolaylıkla uygulanabilmekte
analysis systems, such as the xCELLigence® system, have ve tekrar edilebilmektedir. Ancak gelişen ve değişen teknolojiy-
emerged as a promising substitute for traditional testing methods, le birlikte geleneksel test yöntemlerine bir alternatif olan gerçek
potentially surpassing them in the biocompatibility evaluation zamanlı hücre analiz sistemleri (xCELLigence® sistemi), dental
of dental materials. The xCELLigence® system facilitates the materyallerin biyouyumluluk değerlendirmelerinde tercih edilebi-
concurrent observation and analysis of cells within their authentic lir. Bu sistem ile hücrelerin boyanmasına veya işaretlenmesine
environment, obviating the need for cell staining or marking. This gerek duyulmadan, hücreleri kendi gerçek ortamlarında gerçeğe
review seeks to underscore the advantageous features of the eş zamanlı olarak gözlemlemek ve analiz etmek mümkündür. Bu
xCELLigence® system, which serves to mitigate the drawbacks derleme geleneksel in vitro biyouyumluluk değerlendirme yön-
associated with conventional in vitro biocompatibility evaluation temlerinin dezavantajlarını elimine eden xCELLigence® sisteminin
methods. üstün özelliklerini vurgulamayı amaçlamaktadır.

Keywords: Animals; Biocompatibility; Clinical trials; Cytotoxicity Anahtar Kelimeler: Biyouyumluluk; Diş hekimliği; Hayvanlar; Kli-
tests; Dentistry; Materials testing nik denemeler; Materyal testi; Sitotoksisite testleri

Makale gönderiliş tarihi: 6.12.2023; Yayına kabul tarihi: 2.01.2024

İletişim: Makbule Buse DüNdar Sari
University of Health Sciences, Faculty of Gulhane Dental Medicine, Department of Pediatric Dentistry, Emrah Mah. 06018 Etlik, Keçiören,
Ankara, Turkiye
E-mail: [email protected]
PhD, DDS, University of Health Sciences, Faculty of Gulhane Dental Medicine, Department of Pediatric Dentistry, Ankara, Turkiye

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INTRODUCTION evaluated.9 This biocompatibility evaluation is made

by measuring the viability rate, metabolic functions,
Biocompatibility means that a material does not development rate or other functions of the cells.10
cause tissue reactions such as allergy, local or Tests performed to evaluate the general toxicity of
systemic toxicity, carcinogenic and mutagenic effects materials such as cytotoxicity, carcinogenic effect
when in contact with tissues.1 Biocompatibility may tests, systemic toxicity, inhalation and hemolysis are
vary depending on the type of material, its function, first level in vitro tests.5
the area where it is applied, the monomers in its
structure and the effect of these monomers on cells.2,3 The advantages of in vitro tests are that they are able
to examine a specific function of cell metabolism, are
Negative tissue reactions to non-biocompatible
performed quickly and economically, give quantitati-
materials are interpreted as toxic effects. As a result
ve and comparable findings, are easily standardized
of these reactions, the prevention of synthesis
and reproducible, and have a wider range of use
of various macromolecules and the significant
compared to animal experiments and usage tests.
deterioration in cell structure and functions are called
In addition, there are disadvantages such as using a
cytotoxicity.2
single type of cell for each experiment, culture cells
Before the clinical use of a new material in dentistry, differing from host cells, and the absence of the inf-
the mutagenic, carcinogenic and teratogenic effects lammatory / immune system and circulatory system
of these materials must be examined through that would protect from adverse effects.9,10
comprehensive tests and their biocompatibility must
be evaluated.2,4 If a material is not biocompatible, With in vitro cytotoxicity tests, potential reactions
having superior physical properties is meaningless.5 that may be caused by a material in body tissues
can be imitated and observed in the laboratory en-
The International Organization for Standardization vironment.1
(ISO) documents #7405 and #10993 provide gu-
idance on how to perform biocompatibility tests at The most commonly used in vitro tests for cytotoxi-
certain standards. According to these documents, city evaluations of dental materials are cell cultures.12
tests should be applied in three stages to evaluate
Cell Culture
the biocompatibility of medical materials and devices
used in dentistry. These test methods are respecti- Cell cultures, cell organelles and organ cultures are
vely: biological systems used in cytotoxicity tests. The
most commonly used of these is cell culture.13 The
1- In vitro tests (Phase 1)
main purpose of cell culture applications is the sur-
2- In vivo animal experiments (Phase 2)
vival of cells taken by mechanical and enzymatic
3- Usage tests (Phase 3).6–8
disruption under in vitro conditions, spontaneous
migration from living tissues, and their reproduction
Biocompatibility tests on materials begin with in vitro
by feeding in environments that imitative the body’s
tests using cell cultures. These tests can be applied
unique physiological state and body temperatu-
more easily. In the next stage, animal tests, which
re.2,13–15
are costlier and take longer, are applied. When posi-
tive results are obtained in these tests, more detailed The structure of the cell, its physiological properties,
research can be conducted with usage tests.2 repair and reproduction mechanisms, and pathologi-
cal changes occurring in the cell can be examined
In Vitro Tests
with cell cultures. The effects of materials or drugs
In vitro tests are tests performed outside a living or- on cells can be detected, and structural and chromo-
ganism. In these tests, flasks, test tubes, mamma- somal disorders that may occur as a result of possib-
lian cells in cell culture, tissues, organelles, some le mutagenic effects can be observed.14
enzyme types or bacteria can be used. And the
In cell culture research, two types of cell lines are
response created by the cells as a result of direct/
used: primary cells and continuous cells.2,4,14,15 Pri-
indirect contact with the material to be examined is
mary cells are obtained by taking them directly from

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a living tissue or organ and culturing them for more on requires experience and expertise.
than 24 hours. These cells reflect the physiological • A sterile laboratory environment free of bacterial
state of the tissue. In addition, they show the same and chemical contamination is required.
characteristics as the original tissue cell in terms of • Cells of the desired purity cannot always be obta-
genotype and phenotype.4,13 Subcultures are formed ined.
as a result of primary cell cultures being moved from • It takes time to produce sufficient number of cells.
one culture medium to another after the initial pas- • Freezing cells for a long time causes biochemical
saging process. By performing this process quickly, and genetic changes. This may affect the results of
continuous cell lines are formed.13 the experiment.
• As time passes, the proliferation abilities of the cel-
The use of continuous cell lines in cell culture expe-
ls decrease.
riments to evaluate the biocompatibility of materials
• Since the experiment can be performed with a
is reported to be a more accurate approach in terms
single cell type, information about the effect of the
of standardization. Because primary cell lines have
material on different cell types cannot be obtained
limited reproductive ability and can quickly lose their
with just one experiment.11,17
functions similar to the tissue from which they are
taken. The number of proliferation cycles of conti-
The time the material is in contact is an important fa-
nuous cell lines that undergo transformation is not
ctor for the tests applied. ISO 10993 defines contact
limited, and their metabolic and genetic stability is
periods of less than 24 hours as limited contact, 24
better. In addition, these cells have higher cloning
hours to 30 days as extended contact, and contacts
efficiency, growth rate and tumorigenicity. Persistent
longer than 30 days as continuous contact.6,8 The
cell lines can be easily propagated.11,12,16,17
toxicity of the applied material may vary depending
In most studies investigating the cytotoxic effects of on the density of the material components and the
materials used in dental applications, rat fibroblasts interaction process with the tissue.18,19 Necrosis,
(L929 and 3T3) or human epithelial cells (HeLa) are apoptosis and autophagy develop in cells that are
used as continuous cell lines. Additionally, human exposed to a cytotoxic material for a period of time.
and animal pulp cells, human THP-1 monocytes, As a result of these biological events, cells may lose
and immortalized rat odontoblast cells can also be their viability or proliferation ability.20
used.4,14,16 Due to the homogeneous morphology of
In evaluating the cytotoxicity of dental materials, the
these cells and their reproductive characteristics, it
physical structure of the applied material and its con-
becomes easier to detect in vitro cytotoxicity.1,12
tact with the cell culture are important. This contact
Advantages of cell cultures: can occur directly, indirectly or through the extract of
the biomaterial. In direct contact tests, cells and cul-
• Environmental conditions can be standardized. ture medium are in direct contact. In indirect contact
• It is low cost. tests, there is a permeable barrier between the cells
• Useful in evaluating short-term interactions. and the test materials.12,21 ISO has determined some
• Standard measurements can be made by directly criteria so that tests can be carried out according to
observing the effects on the cell. certain standards. According to ISO 10993-5 criteria,
• It is replicable and results can be obtained faster. in vitro cytotoxicity test methods that can be applied
• The temperature, pH, osmotic pressure, humidity, to dental materials can be listed as follows:2,6,8
oxygen and carbon dioxide amount of the medium
can be controlled.11,17 1. a) Direct cell culture test
i. Direct contact test
Disadvantages of cell cultures: ii. Extract test
b) Barrier test method
• The complex effects of chemical substances can- 2. Agar diffusion test
not be examined. 3. Filter diffusion test
• It cannot provide sufficient information on its own. 4. Dentin barrier test
• Preparation of cultures and microscopic examinati- Direct cell culture test: Dental materials or compo-

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In Vitro Cytotoxicity Tests: xCELLigence® Cilt: 13, Sayı: 2, 2024 Sayfa: 395-401

nents are applied to cells in culture for a short time semble dentin and allow the diffusion of the applied
(less than 24 hours) in the direct contact test. In the material components are used as barriers.6
test performed with this method, the material is in
Agar diffusion test: The agar diffusion test is the
physical contact with the cells or culture medium.
longest-used barrier test method in toxicity experi-
Direct contact of materials and cells, without any
ments. It is a simple and inexpensive method. In this
barrier between them, is essential. Water-soluble
test method, cells stained with neutral dye are cove-
materials can dissolve in the medium and provide
red with agar and the sample material is placed on
successful material-cell contact. For water-insoluble
the agar. Then, the toxicity of the components of the
materials, direct contact can be achieved using dif-
diffusing test materials is examined. Cytotoxicity is
ferent methods. Placing the test sample as close to
evaluated according to the amount of accumulation
the cells as possible, applying it on the cells, placing
of the dye in lysosomes at the end of the 24-hour
it on the bottom of the cell culture container, appl-
incubation period, depending on the permeability of
ying the cell suspension on the sample, or culturing
the cell membrane.6,8,17
the cells by placing them directly on the samples are
some of these methods.13,21,22 Filter diffusion test: In the filter diffusion test, a cel-
lulose acetate filter is placed between the incubated
In the extract test, cytotoxicity evaluation is made by
cells and the material. In order for the cytotoxic effect
contacting the dissolved components of the material
on cells to be observed, the material must diffuse
kept in a liquid solvent with the cells. Serum-conta-
through the filter with pores of 0.45 µm and reach the
ining medium, serum-free medium, physiological
target cells. Damages occurring in the cells are de-
salt solution or one of other suitable solvents can be
termined by examining the staining intensity with a
used as the solvent extraction liquid. The samples
spectrophotometer or by measuring the decoloriza-
are added to the test tubes and the selected extrac-
tion area after staining with neutral red dye.6,8,16,17,23
tion liquid is added to them, then the test tube is left
under the recommended environmental conditions Dentin barrier test: Dentin barrier tests are a comp-
so that the sample material can dissolve and rele- lementary development to cytotoxicity tests and are
ase. At the end of this period, the extracts obtained considered a testing method that can resemble in
are replaced with the medium in the prepared cell vivo conditions. In this method, the diffusion ability
cultures and the cytotoxic effects resulting from the of the monomers of the test material is measured.
experiment are reported.2,6 Sterilized dentin discs obtained from bovine or hu-
man dentin are used as the dentin barrier, and cells
In order to accurately determine the toxic effect of
are placed on one side of the barrier and material is
the material, the extraction liquid must imitate the
placed on the other side.6,17,24,25
clinical use conditions of the material and this en-
vironment must not affect the chemical structure of Animal Experiments
the material. The concentration of the material in
the extract depends on factors such as the volume Animal experiments are performed on experimental
of the extraction liquid, temperature, time, surface animals by imitating the clinical use of dental materi-
area of the material, pH, solubility, diffusion rate and als.5 Mammals such as rats, rabbits and pigs are ge-
osmolarity of the material. 37±2ºC for not less than nerally preferred in these biocompatibility tests. The
24 hours, 50±2ºC for 72±2 hours, 70±2ºC for 24±2 difference between these tests and in vitro cytotoxi-
hours or 121±2ºC for 1±0.2 hours are the extraction city tests is that in vivo systems such as metabolic
environments recommended by ISO.2,6,7 transformation and detoxification can be examined.26

Barrier test method: Dentin in the oral environment Animal tests, which are second level tests, are lo-
acts as a barrier between the pulp and the material cal toxicity tests such as sensitization, subcutaneo-
applied to the cavity. For this reason, tests in whi- us implantation, intraosseous implantation and oral
ch cells come into direct contact with the material mucous membrane irritation tests.5 The effect of
are not sufficient to imitate the clinical situation. In experimental materials placed subcutaneously, int-
the barrier test method, various substances that re- ramuscularly or intrabony on experimental animals

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Dündar Sarı M. B. Cilt: 13, Sayı: 2, 2024 Sayfa: 395-401

is evaluated microscopically and macroscopically the long-term effects of toxic reactions caused by
at different implantation periods (1 week to several the material applied to the cells on cell viability are
months). After the short implantation period (1-2 we- examined. These tests are short-term tests. Although
eks), the level of inflammation around the implanted they are useful, easily applied and rapid tests, they
material is first determined. In the later stages of the do not provide sufficient information to determine the
period, a connective tissue capsule can be obser- long-term toxic effects of the material because they
ved. Thanks to the mucous membrane irritation test, only show dead cells during the test. However, cells
the inflammation caused by the test material in the exposed to toxic effects may need several hours,
mucosa or eroded skin can be examined. Buhler test days or longer to show the consequences of toxicity.
and maximization test are used to detect allergic ef- For this reason, long-term tests are used instead of
fects.4 short-term tests as life evaluation tests.2,22,27

Usage Tests Proliferation Evaluation Tests: Proliferation evaluati-

on tests are one of the oldest and most widely used
Usage tests are third level tests, they are performed
methods in which the effects of various components
by applying dental treatment to experimental ani-
of the experimental material on cell proliferation are
mals or humans.5 Tests performed on humans are
examined. By counting the cells in the cell culture
called ‘clinical trials’. These clinical trials set the gold
after a few days, the effect of the components of the
standard for usage tests.9
material on cell proliferation is determined. Cell coun-
Usage tests are quite complex and costly. When ting at a specific time during the test period does not
long-term effects are investigated, study periods as give a clear result. Therefore, it is necessary to obta-
long as months or years may be required. For clini- in a growth curve in the early stages of testing.2,17,22 A
cal trials conducted in humans, there must be appro- moment in the growth curve should be chosen when
val from government agencies and informed consent the control cells are in the log phase (reproductive
from the patient. There are many legal responsibili- phase) or mid-log phase. When a significant effect is
ties in these tests.9 detected, the obtained improvement curve should be
supported by a second improvement curve or other
Cytotoxicity Evaluation Methods evaluation methods should be applied.2

In the evaluation of cytotoxicity test methods applied Metabolism Evaluation Tests: Metabolism evalua-
according to ISO 7405 and 10993-5 conditions, pa- tion tests have been developed as alternative test
rameters such as cell count, cell membrane dama- methods because the number of samples is large,
ge, staining and metabolic changes are examined. the preparation phase of life evaluation tests takes
The methods determined by ISO to ensure standar- a long time, and test analyzes are time-consuming
dization are as follows:2,6,17 and laborious. It is not possible to directly evaluate
the life of cells with metabolism evaluation tests, but
1- Viability assessment tests
thanks to these tests, ongoing metabolic activity can
2- Life evaluation tests
be detected by determining the increase in the num-
3- Proliferation evaluation tests
ber of cells, DNA or protein synthesis. Through me-
4- Metabolism evaluation tests
tabolism tests and protein content tests, the meta-
bolic capacities of cells are measured to understand
Viability Assessment Tests: With viability assess-
the damage that will occur in the long term.2
ment tests, the proportion of cells that can survive in
cell culture as a result of the short-term toxic effect In metabolism evaluation tests, which are cheap and
of the experimental material is calculated. In these quick methods, the viability of cells is determined
tests, evaluation is made by staining cells with impa- with the help of a spectrophotometer with a micro-
ired and intact cell membrane integrity.2,16,27 plate reader. Lactate dehydrogenase (LDH) test,
alamar blue test and colorimetric MTT test are in-
Life Evaluation Tests: Viability evaluation tests
cluded in this group.2,22
evaluate the colony-forming ability of cells in a low-
density uniform cell suspension.2 With these tests, xCELLigence® System

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In Vitro Cytotoxicity Tests: xCELLigence® Cilt: 13, Sayı: 2, 2024 Sayfa: 395-401

Real-time cell analysis system (RTCA, xCELLi- with the cell density. In addition, data such as cell
gence®) is a system that provides information about index (CI), graph, average value, maximum and min-
cell characterization. Cell proliferation and cytotox- imum values, standard deviation, concentration that
icity can be determined through this system. The produces half the maximum effect (EC50) and half
xCELLigence® system consists of a cell-based mi- concentration of maximum inhibition (IC50) can be
croelectronic cell sensor array that measures the obtained through RTCA software.30
connection or non-connection of cells to electrodes
With the xCELLigence® system, proliferation and
using electrical impedance technology. Electronic
death in cell culture are demonstrated by simulta-
impedance is measured with sensors and changes
neously and continuously detecting impedance (re-
in the electrodes are detected. Cell index is used to
sistance shown by cells to electric current). As the
measure changes in electrical impedance. Electrode
amount of cells adhering to the gold electrodes on
impedance is affected by cell viability, number and
the bottom surface of the E-plates increases, the re-
morphology. The data determined based on the in-
sistance to the current increases, and as it decreas-
crease or decrease in the cell index is evaluated and
es, the resistance decreases. Thanks to this system
finalized by the software.28,29
used in cell culture laboratories, studies such as cell
The xCELLigence® system consists of four main characterization, proliferation and cytotoxicity deter-
components: RTCA analyzer, RTCA single-plate mination, adhesion and receptor-mediated signal
station, RTCA computer with integrated software transmission can be carried out. Additionally, cell
and disposable E-plate 16. This system is used to proliferation and death can be recorded continuously
measure cell viability according to the manufactur- and in real time. The recorded data is transferred to
er’s instructions (Roche Diagnostics GmbH, Mann- the computer screen in graphic form.31
heim, Germany and ACEA Biosciences, Inc., San
The advantages of the xCELLigence® system are
Diego, CA, USA). The RTCA single-plate station fits
that it is less invasive than traditional testing meth-
into a standard tissue culture incubator and mea-
ods, can make easy, simultaneous measurements in
surements are transferred to the computer with a
a shorter time, and provides more reliable results.31
software analyzer. E-plate 16 is a disposable plate
With these new systems that offer real-time analysis,
used to perform cell-based analyzes on the RTCA
cellular data can be received at minimum 15-second
single plate station device. There are gold cell sen-
intervals. Data obtained from the cells in the wells
sor arrays at the bottom of these plates. With E-plate
can be displayed simultaneously on the computer
16, cells in each well can be monitored, experiments
screen, and thus instant changes can be made to
can be performed separately in each well, and their
the experimental protocol, such as stopping the ex-
results can be evaluated separately. Each well on
periment and adding a new substance. Since data
E-plate 16 has a bottom diameter of 5.0 mm ± 0.05
acquisition in the xCELLigence® system is based
mm and a total volume of 243 ± 5 μL. The plate has
solely on impedance measurement, the same cells
a low evaporation lid design. Approximately 80% of
can be reused in another experiment. For example,
the ground area of the wells in the plate is covered
while a material is being examined in real time for
with circular electrodes designed to be used in am-
cytotoxicity, when necessary, the experiment can be
bient conditions between +15 and +40°C, at a maxi-
stopped, cells can be collected from the wells, and
mum relative humidity of 98% without condensation.
its genotoxicity can be evaluated by isolating nucleic
Physiological changes of the cells are detected by
acid. In this way, both time and cost can be saved.30
the electronic impedances detected by the sensor
electrodes. The voltage applied to the electrodes CONCLUSION
during measurement is approximately 20 mV. The
impedance value measured between the electrodes With the development of the product range in dental
in each well varies depending on the ion concen- materials, the number and diversity of tests evalu-
tration in the well, electron geometry and whether ating the biocompatibility of materials have also in-
the cells are connected to the electrodes or not. The creased. The xCELLigence® system as a cell culture
electrode impedance value increases proportionally method for evaluating the biocompatibility of dental

400 ADO Klinik Bilimler Dergisi

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Dündar Sarı M. B. Cilt: 13, Sayı: 2, 2024 Sayfa: 395-401

materials has emerged as a successful alternative to Edit. Tatowa: Humana Press; 2005. p.1–12.
traditional methods. With the xCELLigence® system, 15. Tokur O, Aksoy A. In Vitro Sitotoksisite Testleri. Harran Üniv
cell proliferation and viability levels can be evaluated Vet Fak Derg 2017;6:112–8.
and comments can be made on the biocompatibility 16. Powers J, Sakaguchi R. Craig’s restorative dental materials.
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Real-time cell analysis systems (xCELLigence® sys- 17. Yıldırım ZS, Bakır EP, Bakır Ş, Aydın MS. Diş hekimliğinde
biyouyumluluk ve değerlendirme yöntemleri. Selcuk Dent J
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2017;4:162–9.
evaluations of dental materials in a shorter time, at
a lower cost, and more comprehensively and accu- 18. Saw TY, Cao T, Yap AUJ, Ng MML. Tooth slice organ culture
and established cell line culture models for cytotoxicity assesment
rately. Considering these advantages of the system,
of dental materials. Toxicol Vitr 2005;19:145–54.
the xCELLigence® system can be preferred as a cell
culture method. 19. Mallineni SK, Nuvvula S, Matinlinna JP, Yiu CK, King NM.
Biocompatibility of various dental materials in comtemporary
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