A Critical Appraisal of the Biological

Assessment of Materials in Orthodontics with

Emphasis on the Differences Between
Conventional and 3-D Cell Cultures
Bart Vande Vannet
In this report, we look in a different way at a patient in need for an orthodontic
treatment. Current biomedical literature seeks to link clinical orthodontics with
mainstream molecular-genetic research. The demands of professionalism require the orthodontist to be conversant with all biological principles underlying
treatment. Such knowledge leads to better patient care. (Semin Orthod 2010;
16:274-281.) 2010 Elsevier Inc. All rights reserved.

dverse patient reactions are reported by orthodontists.1 Orthodontic appliances consist of

intraoral metallic and resin-based components,
sometimes combined with extraoral components,
such as face bows or elastics with hooks or clasps.
Leaching of material components from these
appliances is an essential first step in the development of adverse and undesirable patient or
practitioner reactions. For both, they may
present a health hazard.
The risk of adverse effects of an orthodontic
treatment can be defined as the hazard multiplied by the exposure. The risk is the probability
of harm, which concerns both the patient and
the orthodontist. The hazard is related to the
intrinsic properties of a substance, which can be
irritant, corrosive, carcinogenic or even teratogenic. In vitro tests assess the hazard, not the
risk.2 A very good result in vitro after single
application does not guarantee biocompatibility
after repeated or long-term exposure.3
An orthodontic treatment may cause adverse
clinical effects. Clinical use of orthodontic appliances also often is associated with an increase in

Department Orthodontics (SOPA), Vrije Universiteit Brussel

(VUB), Brussels, Belgium.
Address correspondence to B. Vande Vannet, DDS, MSc Orth,
PhD, Department Orthodontics (SOPA), Vrije Universiteit Brussel
(VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail:
bart.vande.vannet@vub.ac.be
2010 Elsevier Inc. All rights reserved.
1073-8746/10/1604-0$30.00/0
doi:10.1053/j.sodo.2010.06.005

274

gingivitis, most often related to poor oral hygiene.4 Intraoral reactions, such as redness, soreness, and swelling of the oral mucosa, gingiva
and/or lips, have been associated with orthodontic appliances. Clinical symptoms may vary
from mild skin and/or mucosal irritations or
allergic reactions to general dermatosis.5 In
some instances, it may be that the appliance or
corrosion products derived from it can cause
local tissue damage, which cannot be distinguished from gingivitis of bacterial etiology.6
The physiological environment of the oral cavity
stimulates the release of components from dental materials.7 For instance, metals, such as
nickel or cobalt, are released from orthodontic
wires. Monomers like bis-GMA [Bowen monomer: 2,2-bis-(4-(2-hydroxy-3-methacryloxypropoxy)
phenyl) propane or bisphenol A diglycidylether
methacrylate] and TEGDMA (triethylene glycol dimetacrylate) leach from the complex
mixtures of orthodontic resin materials. Even
small amounts of these components may have
the potential to alter the integrity of exposed
cells in tissues of the oral cavity.8 Estimation of
cytotoxicity is part of the initial evaluation of
biocompatibility.6
Biocompatibility is the ability of a material to
perform with an appropriate host response in a
specific application.9 The critique against this
definition usually boils down to the fact that it is
not possible to make a single test that determines whether a material is biocompatible. Indeed, because regulation of the immune re-

Seminars in Orthodontics, Vol 16, No 4 (December), 2010: pp 274-281

Differences Between Conventional and 3-D Cell Cultures

sponse and of repair functions in the body is

complicated, it is unlikely that one can rely on a
single test to determine the biocompatibility of
any given material. Sometimes one relies on a
large battery of in vitro tests used in accordance
with the International Organization for Standardization 10993 to determine if a certain material (or
rather biomedical product) is biocompatible.10
These tests cannot be used to determine the biocompatibility of a material, but they comprise an
important step towards the animal testing and
finally clinical trials that will determine the biocompatibility of the material in a given application, and thus biomedical product. In short:
there is no such thing as a universally biocompatible material but there are degrees of biocompatibility.
There are three definitions of biocompatibility:
1. According to Williams definition, defined in
the European Society of Biomaterials consensus conference I,10 it is the ability of a material to perform with an appropriate host response in a specific application.
2. According to the Dorland Medical Dictionary, it
is the quality of not having toxic or injurious
effects on biological systems.11 This definition is not recommended because it only defines biocompatibility as the absence of host
response and does not include any desired or
positive interactions between the host tissue
and the biomaterials.
3. The American Society for Testing and Materials definition is the comparison of the tissue
response produced through the close association of the implanted candidate material to
its implant site within the host animal to the
tissue response recognized and established as
suitable with control materials. The American
Society for Testing and Materials is not recommended because it only refers to local
tissue responses in animal models.
Humans are regularly exposed to a large variety
of foreign substances that are potentially toxic
and harmful to different organs and tissues. Substances capable of producing cell damage are
known as toxins and are classified according to
whether they exert their effects in all individuals,
in a dose-dependent and predictable manner
(intrinsic toxins), or only in some individuals,
usually after several contacts, in a nondose-de-

275

pendent and therefore unpredictable way (idiosyncratic toxins). Intrinsic toxins may act directly
on cellular systems (active toxins) or after biotransformation by hepatocytes, pancreatic cells
or other cells (latent toxins). Biotransformation
is the sum of the processes by which foreign
chemicals are altered by the body, and usually
serves to enhance their detoxification and elimination. However, biotransformation also can result in bioactivation, which involves the production of reactive metabolites that are more toxic,
mutagenic, or carcinogenic than their parent
compound(s).
The balance between detoxification and bioactivation through oxidative phase I reactions
and conjugative phase II reactions often determines the ultimate toxicity of a given compound. More recently, it has become evident
that the pathways for elimination of parent compounds and metabolites through transport
(phase III) also need to be considered. Therefore, knowing the levels, activities, and tissue
distribution of biotransformation enzymes and
transporters in different species is crucial for
understanding the potential health risks upon
exposure to foreign chemicals.11
Idiosyncratic toxicity may be the consequence
of an unusual metabolism of the drug (metabolic idiosyncrasy) or may be mediated by the
immune system after repeated previous contacts.12,13 The toxicity of metal salts and resin
monomers in vitro has been determined as a
basis for the biocompatibility evaluation of dental alloy and composite materials. Currently, employed end points indicate partial or total cell
destruction or inhibition of cell growth. All of
this is assessed on monolayers of human or animal cells.14,15
Given the small amounts of potentially harmful substances released, acute in vivo toxicity is
usually not observed with the clinical use of an
orthodontic device or a dental material. Inflammatory responses of oral tissues are well documented. However, these reactions cannot be easily simulated in a monolayer cell culture. To
simulate the in vivo situation, end points other
that those used to measure acute toxicity may be
more suitable for in vitro testing of dental materials. Human or animal cell lines have been
used for a wide variety of purposes in medical
research, and the number and range available
are increasing. It is necessary, however, to con-

276

Vande Vannet

sider all relevant information regarding the derivation of new materials.

The quality and specificity of the data generated by in vitro models depends on the following
factors16,17:

the use of a biological system that reproduces,

as close as possible the metabolic behavior of
the target organ for the toxic effect of xenobiotics;
the choice of appropriate parameters to evaluate the toxic effects in vitro; and
a correct experimental design so that in vitro
data are predictive for the potential in vivo
effects.

Todays litigious political and social climate and

increased concern about the presence of potentially harmful substances in the environment
and in consumer products requires extensive
testing for any new industrial product that is
brought to the market. A prudent corporation
may spend several million Euros on in vivo procedures, that use animals, such as dogs, cats, and
rodents. To offset some of the in vivo testing
(both in animal and in human), there has been
increasing emphasis on in vitro testing. More
and more, the entire idea of animal testing is
questioned: for instance, the use of animals for
the safety assessment of cosmetics and toothpastes is greatly criticized. Several companies
make abandoning animal testing a sales point.
Consequently, in 1993 the European Union
set up the European Centre for the Validation of
Alternative Methods to support the European
policy to reduce the animal use required for
regulatory testing. In 2003, the European Parliament approved the seventh Amendment to the
European Directive 76/768/EEC that phases
out the use of animals for acute toxicity testing
for all cosmetic products by 2009 and for longterm toxicity by 2013.
In the last decade, a whole spectrum of in
vitro methods has been developed ranging from
simple monolayer cell systems to more complex
three-dimensional tissue engineered models.18
Cell culture techniques have come a long way
since the days when scientists assumed that cells
simply could not survive outside a living organism. Decades of discovery have generated various protocolsincluding reagents and incubation conditions-tailored and attuned to support
the culturing of most types of cells. It was in 1907

that Robert Harrison was the first to dissociate

spinal cord tissues and to place them in clotted
plasma. Incubated in a humidified growth chamber, the nerve cells not only grew and replicated
but also sprouted long axons into the plasma
clot, mirroring the behavior of nerve cells in
vivo. In 1912, Alexis Carrel placed a slice of heart
muscle from a chick embryo in culture medium
and kept the cardiocytes alive for 34 days. Modern tissue culture was born.18
Over the years, researchers have managed to
culture stem cells, genetically engineered cells,
as well as those isolated from plants, insects, and
animals. Proficiency in culturing cells has meant
the harnessing of cells to produce drugs and
develop crops with specific characteristics. In
addition to allowing for greater understanding
of cell physiology and disease development processes, cell cultures are now used directly for
treating patients. Although different cells have
specific needs, most need to be incubated in
growth media, which provide an intricate balance of amino acids, vitamins, salts, and minerals
that work together to keep cells healthy and
proliferating. Using serum-free media allows the
researcher to tightly control the culture and the
option to add only the components necessary for
testing.18
Cell culture models for dental applications
varying from fibroblasts to the present threedimensional models are described in the literature (Table 1).19-43 Table 1 gives an overview of
the present literature since 1987 and describes
methodological reports on organotypic cultures
of human oral epithelium. The listing of these
references indicates methodology and research
areas. The reader is referred to the original articles for details. The information provided also
reflects the variable depth of details. A brief
description of the culture method is followed by
type of medium with specification of serum supplementation (if used). Consequently, they offer
potential utility as a research tool for the study of
different oral cavity processes and for conducting toxicologic experiments but are not always
reliable.44
Cell culture methods for biocompatibility
testing of dental materials have important technical advantages compared with animal experimentation. Usually, in vitro tests are better standardized that in vivo experiments.45-48 Various

277

Differences Between Conventional and 3-D Cell Cultures

Table 1. Methodological Reports on Organotypic Cultures of Human Oral Epithelium* (a review)

State/Origin/Cell
Line

Methods/Culture Conditions

Length of
Study/Longevity Studies/Characteristics of Cell Line

Buccal mucosa and

gingival

De-epidermalized human dermis;

RM medium including 10%
FBS; submerged or air-liquid
interface

15 d or 1-14 d

Buccal mucosa

Explant culture on tissue culture

plastic of gelatin sponge; BEX
medium

2-5-d longevity

Buccal mucosa and

gingival

Contracted collagen lattice

Oral or dermal fibroblasts;
DMEM 10% FBS;
submerged followed by airliquid interface

1 2 wks

Buccal mucosa

De-epidermized human buccal

mucosa or collagen lattice
buccal fibroblasts; F12:DMEM
(3:1) 10% FBS; submerged
followed by air-liquid interface

2 7 Or 7
7d

Buccal mucosa

Collagen lattice buccal

fibroblasts; supplemented
KGM w/o pituitary extract;
submerged by air-liquid
interface

1 10 d

Buccal mucosa

Collagen lattice buccal

fibroblasts; EMHA; submerged
followed by air-liquid interface

2 10 d

Gingiva (junctional
epithelium)

Outgrowth between explant and

high-protein binding
membrane; EMEM 10%
FBS; submerged culture

4, 6 And 8 d

Gingiva

Collagen lattice embryonic

17 d
dermal fibroblasts; DMEM: F12
(3:1) 5% FBS; submerged
culture

Gingiva

Explant culture on decalcified

dentin matrix or w/o filter
separation; EMEM % FBS
Stroma of gingival fibroblasts on
nylon mesh; DMEM 5%
FBS, moist nonsubmerged
culture

10 d

Collegen lattice NIH-3T3

fibroblastst; E-medium;
submerged followed by airliquid interface

7 10 d

Gingiva

Gingiva

3 wks 35 d
longevity

Submerged cells showed

superior terminal
differentiation than air-liquid
interface cells; HPV 16immortalized expressed an
undifferentiated phenotype
Histology comparable with
noncultured tissue; Nnitrosamine metabolism and
tissue binding of reactive
intermediates
Assessment of morphology;
keratinizing vs
nonkeratinizing epithelia;
normal vs delipized serum;
influences of retinoicacid on
TD and keratin expression
Assessment of morphology;
expression of keratins,
growth, basement membrane
and TD markers; influences
of retinoic acid and
calcipotriol; comparisons to
epidermal cells
Expression of keratins, basal
membrane components,
integrins, cell-surface
carbohydrates and wound
healing markers; comparisons
to epidermal cells
Assessment of morphology and
invasiveness; expression of
keratins; conditions
applicable to SV40Timmortalized and carcinoma
cells; multistage model of
carcinogenesis
Epibolus of 5-8 layers formed
between connective tissue of
explant and substratum;
assessment of morphology,
migration and keratins;
comparisons to the in vivo
situation
Assessment of morphology and
keratin expression; comparison
with grafts generated on 3T3
(mouse, Swiss albino,
embryo)-feeder layers
Expression of cell migration,
DNA synthesis, keratins, and
collagenolytic enzyme activity
Assessment of morphology,
viability, and proliferation;
expression of fibronectin,
keratin, basement membrane,
and stromal markers
Assessment of morphology and
filaggrin expression; normal
vs HPV16-immoratlized and
carcinogen-transformed cells;
multistage model of
carcinogenesis

Reference
Sexton et al,
199352

Liu et al,
199353

Kautsky et al,
199535

Chung et al,
199754

Grn et al,
199931

Hansson et al,
200132

Salonen et al,
198941

Gosselin et al,
198928,
199029
Salonen et al,
199155
Odioso et al,
199556

Park et al,
199557

278

Vande Vannet

Table 1. Continued
State/Origin/Cell
Line

Methods/Culture Conditions

Length of
Study/Longevity Studies/Characteristics of Cell Line

Gingiva and other

sites

Contracted collagen lattice

foreskin dermal fibroblasts;
DMEM:F12 (3:1) 5% FBS;
submerged followed by airliquid interface

4 10 d

Gingiva

Collagen lattice foreskin

dermal fibroblasts; DMEM:F12
(3:1) 10% FBS; submerged
followed by air-liquid interface

4-6 7 d

Gingiva

Contracted collagen lattice

foreskin dermal fibroblasts;
transfer to second collagen
lattice; DMEM 5% FBS;
submerged followed by airliquid interface
Polycarbonate filter 3T3
feeder layer; epithelium
generated separately from
fibroblast support (antipodal
culture); submerged followed
by air-liquid interface
Collagen lattice gingival
fibroblasts; KGM; submerged
followed by air-liquid interface

64d

Gingiva

Gingiva

5 11-14 d

2 d 1, 2 or
3 wks

Gingiva
(periodontal
sulcus)

Polyester or collagen membrane;

KSFM elevated Ca2 and
10% FBS; submerged culture

1 wk 1.2 or
3 wks

Gingiva/buccal
mucosa
(nonkeratinizing
epithelia)

Culture on de-epidermalized
human dermis soaked in
collagen; MCDB153 medium
or w/o 10% FBS; submerged
followed by air-liquid interface

4 4, 11 or
18 d; 4 d
1 or 2 wks

Gingiva

Collagen lattice NIH - 3T3

fibroblasts; 1:1 mixture of DKSFM: DMEM/F12 (1:3) 10%
FBS; submerged followed by
air-liquid interface

1 10-14 d

Gingiva

Keratinocytes grown on
3 wks
polyethylene membrane with
agar overlay; fibroblasts grown
on lower surface; FAD with 5%
FBS, submerged culture

Palate (hard)

Culture on de-epidermized
human dermis of human skin;
DMEM: F12 (3:1) 10% FBS;
submerged followed by airliquid interface, the latter
delipidized FBS

2 14 d

Influences of retinoic acid on

differentiation; assessment of
keratin and filaggrin
expression; retroviral
transfection and expression
of retinoic acid receptors;
comparison with epidermal
cells
Assessment of morphology;
expression of keratins and
filaggrin; conditions
applicable to HPV16immortalized cells; model of
carcinogenesis
Model of reepithelialization
and wound healing;
assessment of proliferation
migrations and expression of
TGF- and matrix
metalloproteinase
Assessment of morphology and
keratin expression;
nonsubmerged culture
promotes differentiation;
comparisons to other supports,
ie, collagen lattice and 3T3
Assessment of morphology,
proliferation, keratins and
basement membrane
components; tissue-like
differentiation at later time
points
Assessment of morphology and
keratin expression;
junctional-like or sulcularlike epithelium was induced
dependent on conditions
Assessment of morphology,
proliferation, keratins and
fatty acids; the organotypic
epithelium appeared to be
more active and proliferative
than native keratinized
mucosa
Assessment of morphology and
invasiveness; normal vs
HPV16-immoratlized,
carcinogen-transformed and
carcinoma cells; multistage
modeling of carcinogenesis
Assay standardized by
application of keratinocytes
immortalized by HPV-E6 and
E7; assessment of surface
integrity, proliferation and
keratin expression following
exposure to dental materials
Assessment of morphology and
keratin expression;
conditions with normal
serum preferable to buccal
cells

Reference
Schn and
Rheinwald,
199642

Oda et al,
199658

Garlick et al,
199627

DelcourtHuard et al,
199726

Tomakidi et
al, 1997,
1998,
199944
Papaioannou
et al, 199939

Izumi et al,
1999,
200059,60

Yoo et al,
200047

Tomakidi et
al, 200045

Cho et al,
200024

Differences Between Conventional and 3-D Cell Cultures

279

Table 1. Continued
State/Origin/Cell
Line

Methods/Culture Conditions

Length of
Study/Longevity Studies/Characteristics of Cell Line

Peritonsilar
mucosa and
other sites

Explant culture; EMEM 10%

decalcified FBS

Buccal mucosa

Culture on tr 146 airlifted, living, 5-7 d

multilayered tissue construct,
produced in polycarbonate
inserts in serum-free and
chemically defined medium,
featuring normal ultrastructure
and functionality equivalent to
human epidermis in vivo.

3-4 d

Assessment of histology;
differentiation regulated by
Ca2; a de-stratified explant
with a dorsal layer of basal
keratinocytes can be
redifferentiated
Assessment of histology and
morphology

Reference
Sacks et al,
198540

Vande Vannet
et al,
200649-51

BEX, buccal explants; DK-SFM, serum-free defined keratinocyte medium; DMEM, Dulbeccos modified Eagles medium;
EMHA, epithelial medium with high levels of amino acids; EMEM, Eagles minimum essential medium; FAD, Hams F-12
medium and Dulbeccos modified Eagles medium; FBS, fetal bovine serum; HPV, human papillomavirus; KGM, keratinocyte
growth medium; KSFM, keratinocyte serum-free medium; RM, ; SV40T, simian virus 40 T antigen; TD, terminal differentiation
of the squamous type; TGF-, human transforming growth factor; 3T3 (mouse, Swiss albine, embryo).
*The listing of these references indicates methodology and research areas, and the reader is referred to the original articles
for details. The information provided also reflects the variable depth of details provided by the respective authors.
Listing of the reports is based on site in oral cavity in alphabetical order and year of publication in succession. Priority has
been given to articles from 1987 and onwards because of existing reviews of reports older than 1987.
A brief description of the culture method is followed by type of medium with specification of serum supplementation (if
used). Media abbreviations were used as reported.
The information on length of study often involves separation of the time in submerged culture (first) and air liquid interface
culture (second); time indicating longevity is stated.
On occasion, parts of the information were retrieved from other reports than those listed, e.g., application of the identical
technique for epidermal keratinocytes at earlier date.

in vitro models may address different questions.46

The physical impossibility of directly applying
insoluble materials to a traditional monolayer
culture system had led to significant advances in
the development of three-dimensional culture
systems. At our department we performed the
characterization of epithelial cultures reconstituted from TR146 cells, which were originally
expanded from a squamous cell carcinoma of
human buccal mucosa. Most importantly, these
cells express some keratins of the normal human
buccal mucosa in culture. Multilayered TR146
cells have been reported to model the human
buccal epithelium barrier in vitro.16 However,
this model is not yet validated. In addition, the
characterization of a cell line derived from primary gingival cells is to be performed. There is a
need for both cell lines to be reproducible so
that they could be used for screening.
Furthermore, these three-dimensional tissue
models allow topical application of test materials
and therefore all types of products can be tested
at in use concentrations and at real-time expo-

sures. In other words any type of compound,

whether formulated as a paste, liquid or solid
can now be screened on a batch of human tissues that are identical and that will react in the
same way to the same compound, under the
same experimental conditions.

Acknowledgments
The author would like to thank R. Grafstrm Karolinksa
Institutet, Stockholm, Sweden, for his advice in composing
the table.

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