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Special Issue Reprint

Risk Assessment
of Nanomaterials Toxicity

Edited by
Andrea Hartwig and Christoph van Thriel

www.mdpi.com/journal/nanomaterials
Risk Assessment of Nanomaterials
Toxicity
Risk Assessment of Nanomaterials
Toxicity

Editors
Andrea Hartwig
Christoph van Thriel

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Editors
Andrea Hartwig Christoph van Thriel
Karlsruhe Institute of TU Dortmund
Technology (KIT) Germany
Germany

Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal
Nanomaterials (ISSN 2079-4991) (available at: https://www.mdpi.com/journal/nanomaterials/
special issues/Risk Assessment Nano).

For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Volume Number,
Page Range.

ISBN 978-3-0365-7812-5 (Hbk)

ISBN 978-3-0365-7813-2 (PDF)

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications.
The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND.
Contents

Andrea Hartwig and Christoph van Thriel

Risk Assessment of Nanomaterials Toxicity
Reprinted from: Nanomaterials 2023, 13, 1512, doi:10.3390/nano13091512 . . . . . . . . . . . . . . 1

Claudia Meindl, Markus Absenger-Novak, Ramona Jeitler, Eva Roblegg

and Eleonore Fröhlich
Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine
Release in In Vitro Models of the Respiratory Tract
Reprinted from: Nanomaterials 2023, 13, 682, doi:10.3390/nano13040682 . . . . . . . . . . . . . . . 3

Gabriela de Souza Castro, Wanderson de Souza, Thais Suelen Mello Lima,

Danielle Cabral Bonfim, Jacques Werckmann, Braulio Soares Archanjo, et al.
The Effects of Titanium Dioxide Nanoparticles on Osteoblasts Mineralization: A Comparison
between 2D and 3D Cell Culture Models
Reprinted from: Nanomaterials 2023, 13, 425, doi:10.3390/nano13030425 . . . . . . . . . . . . . . . 23

Elisabeth Elje, Espen Mariussen, Erin McFadden, Maria Dusinska and Elise Rundén-Pran
Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for
Hazard Assessment of Nanomaterials
Reprinted from: Nanomaterials 2023, 13, 407, doi:10.3390/nano13030407 . . . . . . . . . . . . . . . 39

Paul Schumacher, Franziska Fischer, Joachim Sann, Dirk Walter and Andrea Hartwig
Impact of Nano- and Micro-Sized Chromium(III) Particles on Cytotoxicity and Gene Expression
Profiles Related to Genomic Stability in Human Keratinocytes and Alveolar Epithelial Cells
Reprinted from: Nanomaterials 2022, 12, 1294, doi:10.3390/nano12081294 . . . . . . . . . . . . . . 67

Otto Creutzenberg, Helena Oliveira, Lucian Farcal, Dirk Schaudien, Ana Mendes,
Ana Catarina Menezes, et al.
PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of
Graphene-Related 2D Nanomaterials
Reprinted from: Nanomaterials 2022, 12, 1254, doi:10.3390/nano12081254 . . . . . . . . . . . . . . 85

Linda Elberskirch, Adriana Sofranko, Julia Liebing, Norbert Riefler, Kunigunde Binder,
Christian Bonatto Minella, et al.
How Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety
Studies: Evaluation by a Round-Robin Test
Reprinted from: Nanomaterials 2022, 12, 1053, doi:10.3390/nano12071053 . . . . . . . . . . . . . . 119

Mauro Sousa de Almeida, Patricia Taladriz-Blanco, Barbara Drasler, Sandor Balog,

Phattadon Yajan, Alke Petri-Fink and Barbara Rothen-Rutishauser
Cellular Uptake of Silica and Gold Nanoparticles Induces Early Activation of Nuclear Receptor
NR4A1
Reprinted from: Nanomaterials 2022, 12, 690, doi:10.3390/nano12040690 . . . . . . . . . . . . . . . 139

Harald F. Krug
Collection of Controlled Nanosafety Data—The CoCoN-Database, a Tool to Assess
Nanomaterial Hazard
Reprinted from: Nanomaterials 2022, 12, 441, doi:10.3390/nano12030441 . . . . . . . . . . . . . . . 157

Sarah May, Cordula Hirsch, Alexandra Rippl, Alexander Bürkle and Peter Wick
Assessing Genotoxicity of Ten Different Engineered Nanomaterials by the Novel
Semi-Automated FADU Assay and the Alkaline Comet Assay
Reprinted from: Nanomaterials 2022, 12, 220, doi:10.3390/nano12020220 . . . . . . . . . . . . . . . 175

v
Johanna Wall, Didem Ag Seleci, Feranika Schworm, Ronja Neuberger, Martin Link,
Matthias Hufnagel, et al.
Comparison of Metal-Based Nanoparticles and Nanowires: Solubility, Reactivity,
Bioavailability and Cellular Toxicity
Reprinted from: Nanomaterials 2021, 12, 147, doi:10.3390/nano12010147 . . . . . . . . . . . . . . . 199

Sivakumar Murugadoss, Sonja Mülhopt, Silvia Diabaté, Manosij Ghosh,

Hanns-Rudolf Paur, Dieter Stapf, et al.
Agglomeration State of Titanium-Dioxide (TiO2 ) Nanomaterials Influences the Dose Deposition
and Cytotoxic Responses in Human Bronchial Epithelial Cells at the Air-Liquid Interface
Reprinted from: Nanomaterials 2021, 11, 3226, doi:10.3390/nano11123226 . . . . . . . . . . . . . . 219

Sivakumar Murugadoss, Lode Godderis, Manosij Ghosh and Peter H. Hoet

Assessing the Toxicological Relevance of Nanomaterial Agglomerates and Aggregates Using
Realistic Exposure In Vitro
Reprinted from: Nanomaterials 2021, 11, 1793, doi:10.3390/nano11071793 . . . . . . . . . . . . . . 235

Nienke Ruijter, Lya G. Soeteman-Hernández, Marie Carrière, Matthew Boyles,

Polly McLean, Julia Catalán, et al.
The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of
Nanomaterials in the Context of Safe-by-Design
Reprinted from: Nanomaterials 2023, 13, 472, doi:10.3390/nano13030472 . . . . . . . . . . . . . . . 249

vi
 nanomaterials

Editorial
Risk Assessment of Nanomaterials Toxicity
Andrea Hartwig 1 and Christoph van Thriel 2, *

1 Department of Food Chemistry and Toxicology, Institute of Applied Biosciences (IAB), Karlsruhe Institute of
Technology (KIT), Gebäude 50.41 (AVG), Adenauerring 20a, 76131 Karlsruhe, Germany;
[email protected]
2 Leibniz Research Centre for Working Environment and Human Factors, Research Group Neurotoxicology
and Chemosensation, Department of Toxicology, TU Dortmund, Ardey Str. 67, 44139 Dortmund, Germany
* Correspondence: [email protected]

The increasing use of nanomaterials in almost every area of our daily life renders
toxicological risk assessment a major requirement for their safe handling. Thus, risk as-
sessment strategies ensuring the health of individuals exposed to these types of materials
must be adopted and continuously reviewed. Major challenges include the enormous
amount of engineered nanomaterials (ENMs) used in workplaces [1], the limited capacity
for testing ENMs in long-term animal inhalation studies [2], and the political and soci-
etal efforts to reduce animal experiments according to the 3R principles [3]. Against this
background, much attention has been paid to grouping of nanomaterials, mainly based
on their physicochemical properties and their toxicity in various in vitro models. These
new approach methodologies (NAMs) include a detailed characterization of the respective
materials in physiologically relevant media, but also more realistic exposure systems, such
as co-cultures, at the air–liquid interface, combined with comprehensive cellular inves-
tigations providing quite detailed toxicological profiles. These NAM-based approaches
have been recently reviewed by the U.S. Federal Agencies and the authors concluded
that “ . . . two key issues in the usage of NAMs, namely dosimetry and interference/bias
controls, . . . ” are crucial aspects in ongoing validation processes [4]. In workplaces where
inhalation is the major route of exposure, potential toxicity affecting the lungs needs to be
considered. Here, advanced in vitro models have documented their predictive capacity
for adverse outcomes such as lung fibrosis [5]. Neurotoxicity associated with exposure
to nanomaterials is another growing field of scientific investigation [6] and, here, the use
of nanocarriers for drug delivery provides a special “route of exposure” [7]. We initiated
Citation: Hartwig, A.; van Thriel, C.
this Special Issue to further promote scientific progress in the area of nanosafety and are
Risk Assessment of Nanomaterials
glad to share 13 papers on various topics with the readership of Nanomaterials. This Special
Toxicity. Nanomaterials 2023, 13, 1512.
Issue highlights recent advances in the mechanisms of nanomaterial toxicity as well as
https://doi.org/10.3390/
approaches for risk assessment, linking nanoparticle characteristics and in vitro toxicity to
nano13091512
in vivo observations for advanced risk assessment. Here, the availability of data and the
Received: 12 April 2023 development of databases are important.
Accepted: 24 April 2023 With three original articles by Murugadoss, Mülhopt et al., Elje et al., and Meindl
Published: 28 April 2023 et al., addressing various aspects, the respiratory tract toxicities of titanium dioxide, carbon
nanotubes, and nanosilver have been described and some assays can be further validated. A
link between in vitro screening and results from in vivo testing for lung effects is provided
by Creutzenberg et al., describing results from the PLATOX project. Focusing on the aspect
Copyright: © 2023 by the authors.
of data availability and reproducibility, Krug describes the development of the CoCoN-
Licensee MDPI, Basel, Switzerland.
Database, while Elberskirch et al. describe the results of a round-robin test that includes
This article is an open access article
distributed under the terms and
data science tools to increase comparability among different labs. Another relevant and
conditions of the Creative Commons
important aspect is addressed by de Souza Castro et al., comparing 2D and 3D cell culture
Attribution (CC BY) license (https:// models of bone mineralization. Here, the 3D model showed improved induction of bone
creativecommons.org/licenses/by/ osteointegration by nanoparticles. Mechanisms related to the possible genotoxicity of
4.0/). ENM are described in the papers by Schumacher et al., May et al., and Murugadoss, with

Nanomaterials 2023, 13, 1512. https://doi.org/10.3390/nano13091512 https://www.mdpi.com/journal/nanomaterials

1
Nanomaterials 2023, 13, 1512

Godderis et al. also addressing the crucial aspect of realistic exposure scenarios in vitro.
These papers are also relevant to the key issue of dosimetry, as described by Petersen
et al. [4]. The paper by Wall et al. provides new insight into the physico-chemical properties
of particulate and fibrous nanomaterials that can modulate their toxicity. Finally, the review
by Ruijter et al. highlights various aspects of how in vitro methods can be incorporated
into the Safe-by-Design concept that is expected to foster the development of safe ENMs
before they enter the market.

Author Contributions: Writing—original draft preparation, C.v.T.; writing—review and editing, A.H.
and C.v.T. All authors have read and agreed to the published version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Schulte, P.A.; Kuempel, E.D.; Drew, N.M. Characterizing Risk Assessments for the Development of Occupational Exposure Limits
for Engineered Nanomaterials. Regul. Toxicol. Pharmacol. 2018, 95, 207–219. [CrossRef] [PubMed]
2. Landsiedel, R.; Ma-Hock, L.; Hofmann, T.; Wiemann, M.; Strauss, V.; Treumann, S.; Wohlleben, W.; Groters, S.; Wiench, K.; van
Ravenzwaay, B.; et al. Application of Short-Term Inhalation Studies to Assess the Inhalation Toxicity of Nanomaterials. Part. Fibre
Toxicol. 2014, 11, 16. [CrossRef] [PubMed]
3. Caloni, F.; De Angelis, I.; Hartung, T. Replacement of Animal Testing by Integrated Approaches to Testing and Assessment
(IATA): A Call for in Vivitrosi. Arch. Toxicol. 2022, 96, 1935–1950. [CrossRef] [PubMed]
4. Petersen, E.J.; Ceger, P.; Allen, D.G.; Coyle, J.; Derk, R.; Garcia-Reyero, N.; Gordon, J.; Kleinstreuer, N.C.; Matheson, J.; McShan,
D.; et al. U.S. Federal Agency Interests and Key Considerations for New Approach Methodologies for Nanomaterials. ALTEX
2022, 39, 183–206. [CrossRef] [PubMed]
5. Barosova, H.; Maione, A.G.; Septiadi, D.; Sharma, M.; Haeni, L.; Balog, S.; O’Connell, O.; Jackson, G.R.; Brown, D.; Clippinger,
A.J.; et al. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020, 14,
3941–3956. [CrossRef] [PubMed]
6. Boyes, W.K.; van Thriel, C. Neurotoxicology of Nanomaterials. Chem. Res. Toxicol. 2020, 33, 1121–1144. [CrossRef] [PubMed]
7. Mulvihill, J.J.; Cunnane, E.M.; Ross, A.M.; Duskey, J.T.; Tosi, G.; Grabrucker, A.M. Drug Delivery across the Blood-Brain Barrier:
Recent Advances in the Use of Nanocarriers. Nanomedicine 2020, 15, 205–214. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.

2
 nanomaterials

Article
Assessment of Carbon Nanotubes on Barrier Function, Ciliary
Beating Frequency and Cytokine Release in In Vitro Models of
the Respiratory Tract
Claudia Meindl 1 , Markus Absenger-Novak 1 , Ramona Jeitler 2 , Eva Roblegg 2 and Eleonore Fröhlich 1, *

1 Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz, Austria
2 Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences,
University of Graz, Universitaetsplatz 1, 8010 Graz, Austria
* Correspondence: [email protected]; Tel.: +43-31638573011

Abstract: The exposure to inhaled carbon nanotubes (CNT) may have adverse effects on workers
upon chronic exposure. In order to assess the toxicity of inhaled nanoparticles in a physiologically
relevant manner, an air–liquid interface culture of mono and cocultures of respiratory cells and
assessment in reconstructed bronchial and alveolar tissues was used. The effect of CNT4003 reference
particles applied in simulated lung fluid was studied in bronchial (Calu-3 cells, EpiAirway™ and
MucilAir™ tissues) and alveolar (A549 +/−THP-1 and EpiAlveolar™ +/−THP-1) models. Cyto-
toxicity, transepithelial electrical resistance, interleukin 6 and 8 secretion, mucociliary clearance and
ciliary beating frequency were used as readout parameters. With the exception of increased secretion
of interleukin 6 in the EpiAlveolar™ tissues, no adverse effects of CNT4003 particles, applied at
doses corresponding to the maximum estimated lifetime exposure of workers, in the bronchial and
alveolar models were noted, suggesting no marked differences between the models. Since the doses
for whole-life exposure were applied over a shorter time, it is not clear if the interleukin 6 increase in
the EpiAlveolar™ tissues has physiological relevance.

Citation: Meindl, C.; Keywords: carbon nanotubes; toxicity; in vitro models; respiratory tract; bronchial epithelium;
Absenger-Novak, M.; Jeitler, R.;
alveolar epithelium; ciliary beating frequency
Roblegg, E.; Fröhlich, E. Assessment
of Carbon Nanotubes on Barrier
Function, Ciliary Beating Frequency
and Cytokine Release in In Vitro
1. Introduction
Models of the Respiratory Tract.
Nanomaterials 2023, 13, 682. https:// Nanoparticles are used in industry, consumer products, health care and medicine, but
doi.org/10.3390/nano13040682 the safety of some particles is still a matter of discussion [1]. One particle type with unclear
safety is carbon nanotubes (CNTs). CNTs have been evaluated for cytotoxicity, genotoxicity,
Academic Editors: Andrea Hartwig
inflammatory potential and carcinogenicity with variable results [2]. The absorption of
and Christoph Van Thriel
CNTs through epithelial barriers was found to be low. Among all barriers, the respiratory
Received: 21 December 2022 barrier is the less protected and most permeable. The lung consists of the conducting and
Revised: 31 January 2023 the respiratory airways, which have different protection mechanisms and require different
Accepted: 6 February 2023 models for toxicity testing in vitro [3].
Published: 9 February 2023 Reported cytotoxicity of multi-walled CNTs (MWCNTs) varied from 5 ng/mL to
10 mg/mL and based on a systematic review, A549 and HUVEC were the most appropriate
cells for comparison among research groups [4]. The differences in cytotoxicity by a factor
of 106 are thought to be due not only to differences in cells, assays, exposure conditions
Copyright: © 2023 by the authors.
and sample preparation but also to different production of the MWCNTs [4]. To enable
Licensee MDPI, Basel, Switzerland.
better comparison of the results, the use of standardized nanoparticles is suggested. Spon-
This article is an open access article
distributed under the terms and
sored by several European Union Joint programs, European Commission’s Directorate
conditions of the Creative Commons
General Joint Research Centre (JRC) has established the JRC Nanomaterials Repository
Attribution (CC BY) license (https://
of industrially manufactured nanomaterials with the aim of providing the scientific and
creativecommons.org/licenses/by/ regulatory communities with nanomaterials for testing [5]. Standard Operation Procedures
4.0/). for the dispersion of titanium dioxide, silicon dioxide and MWCNTs in 0.05% bovine

Nanomaterials 2023, 13, 682. https://doi.org/10.3390/nano13040682 https://www.mdpi.com/journal/nanomaterials

3
Nanomaterials 2023, 13, 682

serum albumin (BSA) are available due to the NANOGENOTOX European joint action to
exclude differences in the preparation of particle suspensions [6]. The CNT4003 particles,
formerly termed NM-403, belong to the thin and short MCNTs with reported primary
sizes of 12 nm diameter and 443 nm length. They were chosen for this study because
measurements of CNT manufacturing workplaces report sizes of highest exposure between
10 and 100 nm [7–9], whereas the thicker and longer CNTs (e.g., 80 nm × 3.7 μm) form
particles with aerodynamic size of 260–381 nm [10]. No decrease in viability was observed
at 45.7 μg/cm2 NM-403 for A549 alveolar epithelial cells in conventional testing [11].
Air–liquid interface culture (ALI), where cells grow on transwell membranes and are
exposed to medium with nutrients only from the basal side and the apical parts face the air,
is the physiologically relevant culture for respiratory cells. Using this technique, Calu-3
cells, established models for the bronchial epithelium, produce mucus and A549 as models
for the alveolar epithelium surfactant [12,13]. Calu-3 cells also form epithelial barriers
of sufficient tightness identified by transepithelial electrical resistance (TEER) values of
>300 Ω × cm2 . The limitation of Calu-3 cells is that effects of toxicants on ciliary function,
the important clearance mechanism of the upper airways, cannot be assessed because the
cells form only immature and not motile cilia [14]. Reconstructed tissues prepared from
human bronchial epithelial cells, EpiAirway™ from MatTek Cooperation and MucilAir™
from Epithelix Sárl, on the other hand, possess cilia and, therefore, allow the assessment
of this relevant protection system of the lung. The main limitation of A549 cells, which
are routinely used in respiratory toxicity testing, is the fact that despite expression of tight
junction proteins, they do not form a functional epithelial barrier, which corresponds to
the generally low TEER values in the range of 20–60 Ω × cm2 [15,16]. EpiAlveolar™ from
MatTek is composed of alveolar epithelial cells, fibroblasts and endothelial cells and enables
assessment of alveolar barrier function. EpiAlveolar™ enriched with THP-1 macrophages
are also available for more realistic testing of the alveolar barrier [17].
To mimic inhalation of MWCNTs, some researchers have used the VITROCELL
Cloud system [17–19]. According to the producer, 200 μL of nebulization volume are
recommended for the 12-well exposure chambers (https://www.vitrocell.com/inhalation-
toxicology/exposure-systems/vitrocell-cloud-system/vitrocell-cloud-alpha-6 accessed
on 2 May 2021), indicating that each well of a 12-well plate will receive ~17 μL of saline
together with the particles. This solution differs in volume and composition from the lung
lining fluid in the lung. The surface of the lung is covered by a dipalmitoyl-glycero-3-
phosphocholine (DPPC)-rich lung lining fluid of 70–300 nm thickness [20]. The presence
of DPPC is important because nanoparticles in contact with biological fluids will absorb
biomolecules, proteins and lipids onto their surface [21]. This so called “corona” will
determine the cellular response of the host cells. It has been reported in animal studies
that coating with DPPC markedly increased uptake and lung retention of nanoparticles
compared to coating with BSA [22,23]. To take the effect of the DPPC binding into account,
CNT4003 particles were suspended in simulated lung fluid (SLF), a phosphate buffer
containing 0.02% DPPC.
Sauer et al. compared the cytotoxicity of respiratory toxicants in A549 and 3T3 mono-
layers, MucilAir™ and EpiAirway™ tissues [24], but there is no systematic comparison
between models based on cell lines and commercially available reconstructed tissues of
bronchial and respiratory part. This comparison is important because the use of the more
complex reconstructed tissues is associated with higher costs. The aim of this study was to
identify adverse effects of standardized CNTs in physiologically relevant systems and to re-
veal the role of the testing system. To this end, effects of CNT4003 particles were compared
in models of different complexity (monolayers vs. multilayers) and origin (primary vs. cell
lines). The focus is not on cytotoxicity but on functional aspects because physiologically
relevant exposure doses to CNTs are too low to act acutely toxic. For the bronchial part of
the airways Calu-3 cells, EpiAirway™ and MucilAir™ were used. Effects of CNT4003 on
the alveolar part of the airways were studied using A549 cells and EpiAlveolar™ tissues
with and without THP-1 macrophages.

4
Nanomaterials 2023, 13, 682

2. Materials and Methods

2.1. Preparation of Simulated Lung Fluid (SLF)
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Avanti Polar Lipids, Birming-
ham, AL, USA) is dissolved in ethanol and added dropwise to prewarmed (37 ◦ C) phosphate-
buffered saline with Ca2+ and Mg2+ (PBS, ThermoFisher Scientific, Vienna, Austria) to
reach a concentration of 0.02% (w/v) DPPC.

2.2. Preparation of Particle Suspensions

CNT4003 was obtained from the Joint Research Center (JRC, Ispra, Italy). Accord-
ing to the list of representative nanomaterials status June 2016 of the JRC, CNT4003 are
MWCNTs with 189 m2 /g surface area with average length of 443 nm and average diameter
of 12 nm [25]. Suspensions were prepared according to the protocol established in the
NANOGENOTOX project [26] in 0.05% (w/v) BSA and in the same way in SLF to find out if
the same protocol works also for SLF. Briefly, 15.36 mg of CNT4003 powder was prewetted
with 30 μL absolute ethanol. Subsequently, 0.97 mL of SLF or of 0.05% BSA in distilled
water was added while slowly rotating the glass vessel. To prepare the stock solution,
additional 5 mL of SLF or of 0.05% (w/v) BSA was added. Sonication time and amplitude
can be optimized, e.g., 16 min at 10% amplitude or 12 min at 20% amplitude can be used [6].
Suspensions were sonicated on ice with a Branson SFX250 Digital Sonifier (Branson Ultra-
sonic Cooperation, Danbury, USA) equipped with a microtip at 250 W, 20 kHz and 30%
amplitude with 10 s impulses (i.e., 10 s pulse on, 1 s pulse off) for 14 min. Pilot experiments
showed that the use of a higher amplitude in combination with a shorter sonication time
led to a more effective deagglomeration. Suspensions were prepared directly prior to the
administration because it was reported that NM-403 showed high polydispersivity and
were shown to be stable for 1–2 h.

2.3. Determination of Size and Zeta Potential

Measurements were performed at 25 ± 0.5 ◦ C with a particle concentration of 25.6 μg/mL
in triplicates. Hydrodynamic size and polydispersity index (PDI) were measured via
photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments,
Malvern, United Kingdom) equipped with a 532 nm laser. The scattering angle chosen was
173◦ , and the refractive indices for the CNTs and the dispersion medium were 2.50 and
1.330, respectively. The zeta potential was measured by electrophoretic light scattering
(scattering angle of 173◦ ; Zetasizer Nano ZS, Malvern Instruments) considering the same
optical properties of the CNTs and the dispersion media. The calculation was based on the
electrophoretic mobility using Henry’s equation. The zeta potential was −9.9 ± 0.7 mV for
CNT 4003 in BSA and 1.4 ± 0 mV for CNT 4003 in SLF. The hydrodynamic size of particles
suspended in 0.05% BSA determined by PCS was 2327 nm with a PDI of 1.0, indicating that
PCS was not suitable for accurately determining particle size. In addition, SLF contained
DPPS, which formed micelles of 58 ± 7 nm and interfered with the size measurements.
Therefore, the sizes of CNT 4003 were additionally measured by laser diffraction (LD,
Mastersizer 2000, Malvern Instruments, United Kingdom) by adding the CNT suspension
to 18–20 mL SLF or 0.05% BSA until a laser obscuration of 4–6% was achieved. Volume
based values considering d (0.1), d(0.5) and d (0.9) were determined in triplicate at 25 ◦ C
and CNTs showed d(0.1) 974 ± 32, d(0.5) 2254 ± 44 and d(0.9) 5399 ± 71 nm in BSA and
d(0.1) 1290 ± 8, d(0.5) 3829 ± 38 and d(0.9) 6990 ± 43 nm in SLF. These data indicate that
the protocol using SLF leads to a particle distribution similar to that observed with BSA.

2.4. Cellular Models of Bronchial and Alveolar Epithelium

Calu-3 cells were obtained from LGC Standards GmbH (Wesel, Germany) and cultured
in minimum essential medium (MEM), 2 mM L-glutamine, 1% penicillin/streptomycin,
10% fetal bovine serum (FBS) and 1 mM sodium pyruvate. A549 cells obtained from
Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Braunschweig, Ger-
many) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 2 mM L-glutamine,

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1% penicillin/streptomycin (P/S) and 10% FBS. THP-1 was purchased from Cell Line
Services (Eppelheim, Germany) and cultured in RPMI, 2 mM L-glutamine, 1% peni-
cillin/streptomycin and 10% FBS. Cells were passaged at regular intervals.
For the exposures with the CNTs, 0.5 × 106 Calu-3 cells were seeded in 500 μL MEM,
2% L-glutamine and 1% PS + 10% FBS on 12-well polyethylene terephthalate transwell
inserts (pore size 0.4 μm, Greiner Bio-one, Kremsmünster, Austria) with 1500 μL of the
same medium in the basolateral compartment. Medium in the apical compartment was
removed after 24 h, and the medium amount in the basolateral compartment was reduced
to 500 μL, which was changed every 2 or 3 days. Cells were used for the experiments when
they had reached a transepithelial electrical resistance (TEER) value of >300 Ω × cm2 .
A549 was seeded at a density of 0.8 × 105 in DMEM + 10% FBS on 12-well polyethylene
terephthalate transwell inserts (pore size 0.4 μm, Greiner Bio-one) with 1500 μL of the
same medium in the basolateral compartment. Medium in the apical compartment was
removed after 24 h as reported previously [27]. According to these experiments, the ratio
of 9 alveolar cells to 1 alveolar macrophage observed in vivo [28] is obtained by seeding
THP-1 macrophages to the cultured A549 cells in a ratio 1:1. A549 cells were cultured for
9 days in ALI prior to the addition of the THP-1 cells and the average number of A549 cells
was determined in duplicates. For the coculture, RPMI + 10% FBS was added in the basal
compartment and the medium changed every other day.

Differentiation to THP-1 Macrophages for Coculture with A549 Cells or

EpiAlveolar™ Tissues
For coculture with A549 cells, 1.0 × 106 THP-1 cells/mL were seeded in RPMI 1640
containing 10% FBS, 1% L-glutamine and 1% P/S in flasks. Differentiation to macrophages
was induced by addition of 10 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich,
Vienna, Austria) to the media for 48 h. The stimulation medium was changed to medium
without PMA for another 24 h before use in the cocultures. Cells were harvested by
treatment with trypsin/EDTA (0.05%). For addition to EpiAlveolar™ tissues (MatTek
Corporation, Ashland, OR, USA), THP-1 cells were stimulated with 8 nM PMA for 72 h,
harvested by treatment with trypsin/EDTA and added immediately to the tissues.

2.5. Reconstructed Tissues

EpiAirway™ 3D human respiratory epithelial tissues (AIR-100 PC6.5/PE6.5) were ob-
tained from MatTek Corporation. The tissues were cultivated at the air–liquid interface and
medium (MatTek Corporation) changed every other day according to the instructions of
the producer. EpiAlveolar™ 3D tissues with and without THP-1 macrophages (MatTek Co-
operation) were maintained at the air–liquid interface with medium (MatTek Corporation)
changes every other day. Since EpiAlveolar™ 3D tissues with THP-1 macrophages did not
tolerate the long delivery, they were added to the EpiAlveolar™ 3D tissues in the laboratory
in Graz following the protocol of the company in which 25,000 THP-1 macrophages were
added per insert. The cells were seeded in 75 μL media into the apical compartment during
feeding and after 24 h the culture was switched back to air-liquid interface condition.
MucilAir™ tissues were purchased from Epithelix Sárl (Geneva, Switzerland) and
maintained in MucilAir™ culture medium (Epithelix Sárl) in 24-well format Transwell®
cell culture inserts (Sigma-Aldrich) in a humidified incubator (37 ◦ C; 5% v/v CO2 ). The
culture medium was changed every 2–3 days.

2.6. Exposure to Carbon Nanotubes

CNT4003 in different doses were suspended in SLF. A total of 10 μL of the suspension
were used for the 0.336 cm2 tissues and 30 μL for the models with 1.12 cm2 . They were
applied on the surface of the cell monolayers or reconstructed tissues. The entire observa-
tion time was 14 d. After initial addition of the CNT dose for seven days, cell and tissue
surfaces were rinsed with PBS, Transepithelial Electrical Resistance (TEER) measured and
new particles or SLF added for another seven days.

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2.7. Measurement of Transepithelial Electrical Resistance (TEER) Values

TEER values were determined with an EVOM STX-2-electrode (World Precision In-
struments, Berlin, Germany). A total of 0.4 mL MEM was added to the apical and 1.2 mL
MEM to the basolateral compartment for TEER measurements for EpiAirway™ and 0.5 mL
in the apical and 1.5 mL in the basolateral compartment for EpiAlveolar™ tissues. TEER
values were calculated as follows: TEER (Ω × cm2 ) = (Sample–blank resistance, given
in Ω) xmembrane area, given in cm2 . Blank resistance is defined as the resistance of the
membrane without cells. Membrane area is indicated for EpiAirway™ as 0.336 cm2 and for
EpiAlveolar™ as 1.12 cm2 .

2.8. Determination of Viability (MTS Assay)

Viability was assessed at the end of the exposure time (14 d). CellTiter 96® AQueous
Non-Radioactive Cell Proliferation Assay (Promega, Mannheim, Germany) was used ac-
cording to the manufacturer’s instructions. To avoid interference with CNT4003 absorption,
inserts were first rinsed with cell culture medium. Subsequently, 100 μL of the combined
MTS/PMS solution + 500 μL medium was added in each insert. Plates were incubated for
up to 2 h at 37 ◦ C in the cell incubator. The supernatant was transferred into a new plate to
remove remaining CNT4003 particles and absorbance was read at 490 nm on a plate reader
(SPECTRA MAX plus 384, ServoLab, Kumberg, Austria).

2.9. Interleukin Measurements

Medium from the basolateral compartments of all cultures was collected, and the re-
lease of IL-6 and IL-8 was determined using the human IL-6 ELISA set (BD OptEIA™, BD
Biosciences, Heidelberg, Germany) and the human IL-8 ELISA set (BD OptEIA™) according
to the protocol given by the producers. To induce IL-6 and IL-8 secretion, 24 h prior to the
harvesting, 20 μL containing 1, 5 and 10 μg lipopolysaccharide from Escherichia coli 055:B5
(LPS, Sigma-Aldrich) as proinflammatory stimulus was added. The concentration of LPS,
which caused the maximum release of the cytokines by A549 cells was already determined
in a previous study [27]. Absorbance was read at 450 nm (with correction wavelength of
570 nm) on a SPECTROstar (ServoLab) photometer.

2.10. Mucociliary Clearance

Prior to the measurements, tissues were rinsed in PBS and 10 μL of PBS containing
4 × 105 yellow-green, fluorescent carboxyl polystyrene particles (1.0 μm, Thermo Fisher
Scientific, Waltham, MA, USA) was added and migration of the beads monitored for 10–30 s
using Ti2-E/confocal system (Nikon CEE GmbH, Vienna, Austria) equipped with heated
stage (37 ◦ C) and incubation chamber at 10× magnification. Object tracking was performed
by NIS Element software (Nikon CEE GmbH).

2.11. Ciliary Beating Frequency

Prior to the measurements, tissues were rinsed in PBS. Tissues were cut out from the
insert and stripes of epithelium were placed on a glass slide and put into the incubation
chamber (37 ◦ C) on a fully motorized Ti2-E/confocal system (Nikon CEE GmbH) mounted
on an antivibration table. Peripheral and central parts of the stripes were imaged. In pilot
experiments, propranolol hydrochloride (100 μM, Sigma-Aldrich) was included as inhibitor
and 5 μM forskolin (Sigma-Aldrich) + 100 μM 3-Isobutyl-1-methylxanthin (IBMX, Sigma)
as stimulator of CBF. Background noise was determined by measurement of samples fixed
with 4% paraformaldehyde. The background was negligible at 0.15–0.75 Hz. Further,
the reaction to 1, 5 and 10 μg/insert LPS was recorded. Specimens were examined using
20× magnification. Beating ciliated edges were recorded using a digital high-speed video
camera (Andor Zyla VSC-08691, Oxford Instruments, Wiesbaden, Germany) at a rate of
100 frames per second with a frame size of 512 × 512 pixel. Five regions of interest (ROI)
were analysed per sample. CBF was calculated by NIS Element software (Nikon CEE

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GmbH) and indicated as beats/second (Hz). In the later experiments, only propranolol
was used because no prominent increase in CBF was seen.

2.12. Histology
MatTek tissues were fixed in 4% paraformaldehyde and embedded in paraffin us-
ing Tissue-Tek® VIP™ 5 (SanovaPharma GesmbH, Gallspach, Austria). Radial sections
of 2–5 μm were cut at a rotary microtome, stained with hemalaun and viewed with an
Olympus BX51 microscope. Additional sections were cut for immunocytochemical staining.

2.12.1. Immunocytochemical Detection of α-Tubulin

Sections were incubated with DakoCytomation Target Retrieval Solution pH 6.0 (Ag-
ilent, Vienna Austria) in decloaking chamber (Biocare Medical, Pacheco, CA, USA) for
10 min at 110 ◦ C and 20 s at 85 ◦ C. Sections remained in the solution for cool down for
30 min. Three rinses with PBS were performed prior to the incubations with blocking
serum (1% goat serum in PBS for 30 min at RT) and between each staining step. Antibodies
were diluted in antibody diluent (DAKOCytomation, Hamburg, Germany). Incubation
with anti-α-tubulin antibody (mouse, 1:500, Thermo Fisher Scientific) or control mouse IgG
(negative control, Linaris, Mannheim, Germany) for 1 h at RT was followed by incubation
with anti-mouse Alexa 488 antibody (goat, 1:400, Thermo Fisher Scientific) in antibody dilu-
ent (DAKOCytomation) for 30 min at RT and counterstain with Hoechst 33342 (1 μg/mL,
Thermo Fisher Scientific) for 15 min at RT. After three final washes in PBS, sections were
mounted with fluorescence mounting media.

2.12.2. Immunocytochemical Detection of Mucin

Antigen retrieval was performed with DakoCytomation Target Retrieval Solution pH
9.0 (DAKO) in decloaking chamber (Biocare Medical) for 10 min at 110 ◦ C and 20 s at 85 ◦ C.
Sections remained in the solution for cool down for 30 min. Three rinses with PBS were
performed, cells permeabilized with PBS plus 0.2% Triton X-100 for 2 h at RT. Unspecific
antibody binding was blocked with 10% normal goat serum for 30 min at RT. Incubation
with anti-mucin 5AC (mouse, 1:200, Abcam, Cambridge, United Kingdom) antibody was
performed for 1 h at 37 ◦ C, incubation with anti-mouse Alexa 488 antibody (goat, 1:400,
Thermo Fisher Scientific) for 1 h at 37 ◦ C and with Hoechst 33342 (1 μg/mL, Thermo Fisher
Scientific) in PBS for 15 min at RT.

2.12.3. Immunocytochemical Detection of Macrophages

After deparaffinization, cells were permeabilized by incubation with 0.1% Triton X-
100 in PBS for 60 min at RT and incubation with APC-CY7 anti-CD45 (mouse, 1:500, BD
Biosciences, Vienna, Austria) for 1 h at RT and counterstain of nuclei with Hoechst 33342
(1 μg/mL, Thermo Fisher Scientific) for 30 min at RT.

2.12.4. Staining of Whole Mounts with Phalloidin

EpiAlveolar™ tissues were fixed for 15 min with 4% paraformaldehyde. After three
washes with PBS, the tissues were permeabilized with 0.1%Triton X100 in PBS, washed
three times in PBS again and subsequently incubated with Alexa Fluor™ 488 Phalloidin
(Thermo Fisher Scientific, 1:100 in antibody diluent) and Hoechst 33342 (1 μg/mL, Thermo
Fisher Scientific) for 30 min at RT.
Images were taken at a Ti2-E/confocal system (Nikon CEE GmbH) with ex/em of
395 nm/414–450 nm for Hoechst 33342 and 470 nm/500–530 nm for α-tubulin, mucin 5AC
and phalloidin, and 640 nm/660–850 nm for CD45.

2.13. Statistics
Experiments were performed in triplicates and repeated at least two times. Data from
all experiments were analyzed with one-way analysis of variance (ANOVA) followed by

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Tukey’s HSD post hoc test for multiple comparisons (SPSS 28 software). Results with
p-values < 0.05 were considered to be statistically significant.

3. Results
In all evaluations, CNT4003 was applied in SLF and the following parameters and
assays used as readout: MTS assay for cytotoxicity, TEER measurements for epithelial
barrier tightness, interleukin 6 and 8 levels for proinflammatory effects, movement of
polystyrene marker particles for mucociliary clearance and high-speed video microscopy
for ciliary beating frequency. IL-6 is a marker for acute and chronic inflammation and
IL-8 a marker for acute inflammation and a chemoattractant for neutrophils [18]. Doses
are indicated as μg/insert and the distribution of CNT4003 on the cell-grown inserts
documented. After 14 d of exposure, several CNT4003 agglomerates were visible in the
central region of inserts containing mucus-producing cells and rare agglomerates in the
inserts containing EpiAlveolar™ tissues (Figure S1, Supplementary Material). This can be
explained by the fact that mucus can trap the agglomerates better than surfactant.

3.1. Particle Effect in Models of the Bronchial Part of the Respiratory Tract
Calu-3 cells, EpiAirway™ and MucilAir™ tissues were used as models for the bronchial
part and A549 and EpiAlveolar™ tissues with and without macrophages as models for
the alveolar part. The models were characterized regarding the stability and ability to
react to LPS as an inflammatory stimulus. Evaluation of CNT4003 effects over 14 d cor-
responded to the recommendation of Behrsing et al. for analysis in EpiAirway™s and
MucilAir™ tissues [29].

3.1.1. Calu-3 Cells

No cytotoxicity of CNT4003 was seen up to 50 μg with a viability of 86 ± 15% of
untreated controls (Figure S2, Supplementary Material).
As seen in the immunocytochemical staining, Calu-3 monolayers do not form cilia but
contain mucus-producing cells (Figure 1). Alpha tubulin is a microtubule marker, and the
tubulin-dynein system is a central part of flagellar and ciliary movement [30]. To a lesser
extent, tubulin is also expressed in the cytoplasm for structural support, a pathway for
transport and force generation in cell division [31].

Figure 1. Immunochemical detection of tubulin (A) and identification of mucus-producing cells by

anti-mucin 5AC staining (B). Tubulin is contained in high amounts in cilia and in lower amounts in
the cytoskeleton. In the absence of cilia, the staining of the cytoskeleton and of immature cilia (arrow)
is visible. Scale bar: 50 μm.

Stability of the Calu-3 model has been shown previously [32]. In this study, TEER
values decreased significantly from 1 d to 14 d of culture in all cultures with no difference
between untreated and CNT4003-treated cultures (Figure 2). The decrease upon LPS
treatment at 7 d was significant compared to 1 d and also significantly more pronounced
than that of the control.

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Figure 2. Time-dependent changes of transepithelial electrical resistance (TEER) in Calu-3 cells

exposed to simulated lung fluid (SLF) alone, LPS or SLF containing CNT4003. Significant changes
compared to 1 d are indicated by an asterisk and significant differences between treated and control
cells by a hash.

Stimulation with 10 μg LPS increased IL-6 and IL-8 secretions at all time points
significantly (Table 1). Interleukin-6 levels of cultures treated with 25 μg CNT4003 after one
and seven days were significantly lower than the levels of the controls (Table 1). Interleukin
8 levels were significantly increased after one day and decreased after seven days. No
changes were seen after 14 days.

Table 1. Time-dependent changes (%) of cytokine secretion by Calu-3 upon stimulation with 10 μg
LPS or 25 μg CNT4003. Secretion of unstimulated tissues is set as 100%. Significant differences
(p < 0.05) between treated and untreated samples are indicated by an asterisk.

Cytokine Day 1 Day 7 Day 14

Interleukin 6 LPS 478 ± 71 * 703 ± 9 * 722 ± 97 *
Interleukin 8 LPS 1504 ± 69 * 367 ± 88 * 266 ± 32 *
Interleukin 6 CNT4003 55 ± 38 * 25 ± 38 * 116 ± 36
Interleukin 8 CNT4003 118 ± 6* 32 ± 4 * 109 ± 2

3.1.2. EpiAirway™ Tissues

EpiAirway™ cultures are composed of columnar epithelium cells arranged as three
to five rows of nuclei (Figure 3A). Cilia at the apical surface are seen at low density. The
composition of the epithelium of bronchial epithelial cells and goblet cells is identified by
anti-α-tubulin staining (Figure 3B) and anti-mucin 5AC staining (Figure 3C), respectively.
TEER values of EpiAirway™ tissues over 28 d were lower than in the other models
of the bronchial tract but remained constant for 3 weeks with 259 ± 124 Ω × cm2 at 1 d
and 244 ± 69 Ω × cm2 at 21 d. Only after 28 d, a significant decline to 110 ± 67 Ω × cm2
was seen.
CNT4003 particles did not affect TEER values over the entire observation time of 14 d,
whereas the initially higher TEER values of the cultures exposed to LPS were significantly
decreased compared to 1 d at 7 d and 14 d (Figure 4).

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Figure 3. Morphology of EpiAirway™ tissues according to hemalaun staining (A) and cellular
composition according to immunochistochemical staining. Scale bar: 100 μm. Anti-α-tubulin (B) and
anti-mucin 5AC (C) staining (green) for visualization of bronchial epithelial and goblet cells. Controls
with mouse IgG instead of primary antibody are shown on the right side. Nuclei are counterstained
with Hoechst 33342 (blue). Scale bar 50 μm.

Figure 4. Time-dependent changes of transepithelial electrical resistance (TEER) in EpiAirway™

tissues exposed to simulated lung fluid (SLF) alone or SLF containing CNT4003. Significant decreases
compared to 1d are indicated by an asterisk.

EpiAirway™ tissues reacted to stimulation with all concentrations of LPS with signifi-
ant increases of IL-6 and IL-8 secretions compared to unstimulated tissues at all time points
with the exception of IL-6 at day 7 (Table 2).
For assessment of the clearance function of the bronchial epithelium, mucociliary
clearance and CBF were evaluated. No transport of the polystyrene indicator beads was
detected in the EpiAirway™ tissues, presumably due to the low density of the cilia.

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Table 2. Time-dependent changes (%) of cytokine secretion by EpiAirway™ tissues upon stimulation
to 1, 5 and 10 μg lipopolysaccharide (LPS). Secretion of unstimulated tissues is set as 100%. Significant
differences (p < 0.05) between treated and untreated samples are indicated by an asterisk.

Cytokine LPS Day 1 Day 7 Day 14

1 μg 228 ± 83 * 89 ± 7 325 ± 18 *
Interleukin 6 5 μg 368 ± 165 * 163 ± 7 * 211 ± 18 *
10 μg 847 ± 327 * 240 ± 12 * 383 ± 24 *
1 μg 133 ± 10 * 255 ± 46 * 291 ± 37 *
Interleukin 8 5 μg 298 ± 18 * 262 ± 22 * 329 ± 26 *
10 μg 509 ± 12 * 595 ± 7 * 428 ± 4 *

Prior to the assessment of CNT4003, the reaction to LPS was tested. It was found
that exposure to LPS (1, 5, 10 μg) increased CBF at all concentrations significantly from
9.9 ± 1.7 Hz (untreated controls) to 15.3–17.8 Hz at 7 d and from 12.7 ± 1.4 Hz (untreated
controls) to 15.1–16.2 Hz at 14 d. Basal CBF was significantly lower in this batch of tissues
than the frequencies measured in the batch where CNT4003 were tested and no LPS
was included.
CBF of untreated tissues was highest at 1 d and significantly lower at all time points
(Table 3). The positive control propranolol decreased CBF significantly at all time points.
Significant decreases of CBF upon exposure to CNT4003 compared to the respective
medium control were not noted. There was a trend of higher CBF in the peripheral
regions (15.8 ± 4.2 Hz) than in the central regions of the insert (14.7 ± 2.8 Hz).

Table 3. Ciliary beating frequency (Hz) in EpiAirway™ tissues after exposure to negative control,
positive control (propranolol) or CNT4003. Significant differences (p < 0.05) between treated and
untreated samples are indicated by an asterisk. Significant changes over time of the untreated tissues
are marked by a hash.

Treatment Day 1 Day 7 Day 14

Medium 34 ± 6 27 ± 2 # 23 ± 8 #
Propranolol 4±0* 1±0* 0±0*
CNT4003 23 ± 10 22 ± 4 16 ± 2

3.1.3. MucilAir™
MucilAir™ tissues are composed of two to three rows of cells. Cilia are already visible
in the hemalaun staining (Figure 5A). They form much longer structures at the apical surface
(Figure 5B) than in the EpiAirway™ tissue (Figure 3B). Further, rare mucin-producing cells
can be seen in the MucilAir™ tissues (Figure 5C).
TEER values of the untreated MucilAir™ cultures significantly decreased over 21 d
for cultures from 563 ± 66 Ω × cm2 at 1 d to 282 ± 24 Ω × cm2 at 21 d. Therefore, tissues
were studied for only 14 d of exposure with LPS and CNT4003. While TEER values of the
LPS-treated tissues were significantly decreased compared to 1 d, CNT4003 particles did
not affect TEER values over the entire observation time of 14 d (Figure 6).
The reactivity of the tissues to an inflammatory stimulus was tested by incubation with
1, 5 and 10 μg LPS at 1 d, 7 d and 14 d and quantification of IL-6 and IL-8 was performed.
IL-6 levels in LPS-stimulated cultures at all time points and all LPS concentrations were
significantly lower than that of the untreated controls (Table 4), which is consistent with
other studies reporting low IL-6 levels in LPS-stimulated MucilAir™ tissues. In contrast to
the action of IL-6, a robust increase in IL-8 secretion was seen after stimulation with LPS at
all concentrations. Metz et al. suggested that concentrations of 10 μg/mL LPS were too
low to induce a significant increase in IL-6 [33]. IL-6 secretions of the CNT4003-stimulated
MucilAir™ tissues were significantly lower than the unstimulated tissues at all time points

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(Table 4). IL-8 secretions were also significantly lower than that of the unstimulated controls,
except at 1 d.

Figure 5. Morphology of MucilAir™ tissues according to hemalaun staining (A, scale bar: 100 μm)
and cellular composition of the tissues according to immunocytochemistry (B,C). Anti-α-tubulin
staining (green, (B)) visualized cilia of bronchial epithelial cells and anti-mucin 5AC staining (green,
(C)) visualized goblet cells. Nuclei are counterstained with Hoechst 33342 (blue). Scale bar 50 μm.

Figure 6. Time-dependent changes in TEER values of MucilAir™ tissues exposed to simulated

lung fluid (SLF) alone or with SLF containing CNT4003. Significant decreases compared to 1 d are
indicated by an asterisk.

MucilAir™ tissues were able to transport the polystyrene marker beads. However,
since there was no coordinated beating like in the in vivo situation, there was no linear
transport of the beads. If transport was seen, the beads moved in circles (Video S1, Supple-
mentary Material). Quantification of the effect of the CNT4003 particles was not possible
because the diameter and number of the vortexes showed prominent variations between the
tissues. The diameter had an influence on the velocity because transport at the periphery
of the vortex was faster than at the center.
CBF of untreated tissues was significantly increased at 14 d compared to 1 d (Table 5).
Propranolol treatment decreased CBF significantly at all time points. Additionally, the
treatment with 10 μg LPS decreased CBF significantly at 14 d. There was no difference
between central (19.5 ± 7.1 Hz) and peripheral regions of the insert (20.6 ± 6.0 Hz).
CNT4003 exposure did not changes CBF to significant degree.

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Table 4. Time-dependent changes (%) of cytokine secretion by MucilAir™ tissues upon stimulation
with 1, 5 and 10 μg lipopolysaccharide (LPS) or CNT4003. Secretion of unstimulated tissues is set as
100%. Significant differences (p < 0.05) between treated and untreated samples are indicated by an
asterisk. Abbreviation: n.a., not available.

Cytokine Stimulus Day 1 Day 7 Day 14

LPS_1 μg 69 ± 2 * n.a. a 44 ± 3 *
Interleukin 6 LPS_5 μg 77 ± 17 * n.a. a 74 ± 15 *
LPS_10 μg 52 ± 3 * n.a. a 83 ± 5 *
LPS_1 μg 164 ± 2 * 214 ± 172 * 250 ± 142 *
Interleukin 8 LPS_5 μg 143 ± 2 * 470 ± 131 * 477 ± 37 *
LPS_10 μg 277 ± 161 * 443 ± 132 * 659 ± 26 *
Interleukin 6 CNT4003 43 ± 42 * 60 ± 26 * 78 ± 26 *
Interleukin 8 CNT4003 54 ± 12 * 108 ± 13 80 ± 17
a values deleted due to assay interference.

Table 5. Ciliary beating frequency (Hz) in MucilAir™ tissues after exposure to control, lipopolysac-
charide (LPS) or 25 μg CNT4003. Significant (p < 0.05) differences in the medium-treated group at
different time points are indicated by an asterisk and differences of the treatment to the medium
control with a hash.

Treatment Day 1 Day 7 Day 14

Medium 15 ± 5 21 ± 6 28 ± 3 #
Propranolol 5±1* 5±1* 8±4*
CNT4003 12 ± 2 18 ± 8 23 ± 7
10 μg LPS 14 ± 3 19 ± 6 19 ± 4 *

3.2. Assessment of Effects in Models for the Alveolar Part of the Respiratory Tract
3.2.1. A549 Monocultures and Coculture with THP-1 Macrophages
Exposure to 12.5 and 25 μg CNT4003 had no negative effect on the viability of A549
cells in monoculture and coculture with THP-1, but exposure to 50 μg CNT decreased
viability significantly in both types of cultures (Figure S3, Supplementary Material).
A549 cells in mono and coculture with THP-1 macrophages secrete low basal levels of
IL-6 (A549 monoculture; 0–1.4 pg/mL and 0.04–0.1 pg/mL in coculture; ref. [27]) and were
below the limit of detection in this study. They react, however, at lower concentrations than
the bronchial models to LPS and, upon stimulation with LPS, IL-6 levels between 140 and
279 pg/mL in A549 monocultures and between 2 and 3 ng/mL in cocultures with THP-1
were measured. IL-8 levels were 21–27 ng/mL and 53–84 ng/mL, respectively (Table 6).
This indicates that the cocultures produced approximately 10 times higher amounts of IL-6
and 4 times higher levels of IL-8 upon LPS stimulation than the A549 monocultures. In
contrast to LPS, exposure to 25 μg CNT4003 did not stimulate the secretion of IL-6 and IL-8,
which remained below the detection limit.

Table 6. Time-dependent changes in interleukin 6 secretion (pg/mL) and interleukin 8 (ng/mL) secre-
tion by A549 mono and A549/THP-1 coculture upon stimulation with 100 ng/mL lipopolysaccharide
(LPS). Since cytokine levels of untreated cultures were zero or below zero, normalization to % of
control could not be made.

Cytokine Day 1 Day 7 Day 14

Interleukin 6_A549 140 ± 33 279 ± 91 182 ± 48
Interleukin 8_A549 21 ± 7 27 ± 9 20 ± 2
Interleukin 6_A549/THP-1 2025 ± 152 2137 ± 587 3195 ± 93
Interleukin 8_A549/THP-1 81 ± 11 53 ± 29 84 ± 20

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3.2.2. EpiAlveolar™ Tissues with and without THP-1 Macrophages

EpiAlveolar™ tissues consist of alveolar epithelial cells and fibroblasts at the apical
site and endothelial cells at the basal site of the membrane [17]. In the hemalaun-stained
sections, three to four rows of cells can be seen and tissues with THP-1 (Figure 7B) appear
thicker than those without THP-1 (Figure 7A). In tissues with THP-1 cells, the macrophages
were identified by CD45-immunoreactivity (Figure 7C).

Figure 7. Radial section of EpiAlveolar™ tissues without THP-1 (A) and with THP-1 (B) macrophages.
Endothelial cells at the basal side of the transwell membrane are visible in B (arrow). (C) CD45-
immunoreactive THP-1 macrophages are rarely seen (arrowhead). Scale bar 50 μm.

Exposure to 25 μg CNT4003 particles did not decrease viability in EpiAlveolar™

tissues with and without THP-1 cells (Figure S4, Supplementary Material).
EpiAlveolar™ tissues formed tight epithelial barriers with TEER values > 850 Ω × cm2
(Figure 8). TEER values of LPS-exposed tissues reached 1260 Ω × cm2 in the tissues with
THP-1 and 1099 Ω × cm2 in the tissues without THP-1. There were no differences between
tissues with and without THP-1 macrophages and CNT4003 treatment did not influence
barrier tightness. At 14 d, prominent variations between tissue replicates (328–974 Ω × cm2
in tissues with THP-1 and 322–1701 Ω × cm2 in tissues without THP-1) were noted. TEER
values may be a good parameter to screen the quality of the tissues because after one
delayed delivery, TEER values of the tissues were 97 ± 137 Ω × cm2 .
EpiAlveolar™ tissues reacted to stimulation with 1–10 μg LPS with variable and mod-
erate increases in IL-6 and IL-8 secretion. Significant increases in cytokine secretion were
mainly seen after stimulation with 5 and 10 μg LPS (Supplementary Material Table S1). In
general, more stimulations with significant increases in IL-6 and IL-8 were seen in tissues
without THP-1. Treatment with CNT4003 induced a significant increase in IL-6 secretion
in EpiAlveolar™ tissues with and without THP-1 macrophages (Table 7). The response in
the tissues without THP-1 macrophages was significantly higher than in the tissues with
THP-1 cells.

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Nanomaterials 2023, 13, 682

Figure 8. TEER values of EpiAlveolar™ tissues with and without THP-1 treated with control (SLF) or
12.5 and 25 μg CNT4003 particles at 7 d.

Table 7. Time-dependent changes (in %) of cytokine secretion by EpiAlveolar™ tissues without THP-
1 (Epi) and with THP-1 (Epi/THP-1) upon stimulation with lipopolysaccharide (LPS) and CNT4003.
Secretion of unstimulated tissues is set as 100%. Significant increases (p < 0.05) between treated and
untreated samples are indicated by an asterisk and significant decreases by a paragraph sign.

Cytokine LPS_1 μg LPS_5 μg LPS_10 μg CNT_12.5 μg CNT_25 μg

Interleukin 6_Epi 21 ± 1 § 112 ± 2 174 ± 7 * 750 ± 244 * 618 ± 72 *
Interleukin 8_Epi 102 ± 12 327 ± 36 * 305 ± 41 * 82 ± 42 93 ± 21
Interleukin 6_
65 ± 30 104 ± 30 214 ± 48 * 288 ± 78 * 390 ± 139 *
Epi/THP-1
Interleukin 8_
100 ± 4 107 ± 81 883 ± 29 * 174 ± 53 102 ± 40
Epi/THP-1

4. Discussion
The tightness of the epithelial barrier and low basal levels of proinflammatory cy-
tokines, are crucial properties of the healthy lung, and the used models should be able to
assess these parameters. In this comprehensive study, CNTs were evaluated for potential
adverse effects on the bronchial and alveolar part of the respiratory system. Further, not
only acute cytotoxicity but also epithelial barrier properties, release of cytokines and CBF
were used as readout parameters.

4.1. Bronchial Models

The suitability of Calu-3 cells as a model for the respiratory barrier and identifi-
cation of inflammatory effects has already been reported. In this study, TEER values
were 210–365 Ω × cm2 , which is within the range (~100 Ω × cm2 , ~250 Ω × cm2 and
390 Ω × cm2 ) reported in the literature [34–36]. Variations in TEER values of EpiAirway™
tissues can show prominent interdonor variations from ~300 Ω × cm2 to >900 Ω × cm2 [34]
and values obtained in this study were at the lower end of this range. MucilAir™ tissues in
this study showed initial TEER values of 548 ± 65 Ω × cm2 . Similar to the EpiAirway™
tissues, there were prominent variations depending on the batch ranging from 346 ± 14 to
638 ± 81 Ω × cm2 [37]. The difference between different batches of EpiAirway™ tissues
suggests differences between donors or prolonged delivery conditions.

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Nanomaterials 2023, 13, 682

Similar to the values in this study, IL-6 and IL-8 values in EpiAirway™ tissues were
reported as 100–350 pg/mL and 14,500 ± 3000 pg/mL, respectively [8], and 91 ± 40 pg/mL
IL-6 and 5300 ± 220 pg/mL IL-8 [36]. Interbatch levels of IL-6 and IL-8 secretion in
MucilAir™ can vary by a factor of two to four (IL-6: 58 ± 26–205 ± 130 pg/mL and
IL-8: 4.14 ± 0.61–8.19 ± 3.29 ng/mL) [37]. MucilAir™ tissues in this study secreted up to
358 pg/mL IL-6, while IL-8 were around 100 times higher. The much lower IL-6 than IL-8
secretion by MucilAir™ tissues has also been observed in other studies (100 pg/mL vs.
10,000 pg/mL; [38]). Therefore, most researchers used only IL-8 with basal secretions of
~20 ng/mL, 1.8–5.2 ng/mL and 5–30 ng/mL as indicator for inflammation [39–41].
Normal CBF in human bronchi was reported as 12–15 Hz [42], but great variation
from 4 to 19 Hz were reported for human conducting airways [43]. For bronchial epithelial
cells of EpiAirway™, a basal CBF of ~18 Hz (17.62–18.02 Hz) has been reported [44], which
is slightly lower than the frequencies determined in this study. MucilAir™ tissues in this
study had higher CBF than EpiAirway™ tissues. The different ratio of cilia-bearing to
mucus-producing cells between the tissues may be the reason for this difference. The
average CBF of MucilAir™ tissues in the literature are indicated as 15.8 ± 0.3 Hz and
11.7 ± 1.2 Hz [43,45], and Beyeler et al. observed higher CBF in the peripheral than in the
central region [18]. This trend was also to some extent seen in the EpiAirway™ tissues
but not in the MucilAir™ tissues of this study. It indicates that it might be good to use
similar regions of the insert for the analysis and indicate that in the protocol, e.g., Kim et al.
measured 1–2 mm away from the center of the insert [46]. While propranolol reduced CBF,
no marked increase in CBF upon administration of forskolin to the tissues was seen. The
lack of a strong increase of CBF was also observed in mouse and rat lung slices [47,48]. The
authors hypothesized that cells were preactivated by the mechanical manipulation and
beat at their maximum frequency. A decrease of CBF has been observed as a reaction to
bacteria and diesel exhaust particles and can be interpreted as a toxic response [42]. In this
study, 10 μg LPS decreased CBF in the MucilAir™ tissues but increased it in EpiAirway™
tissues. The different reaction may be due to the different basal CBF, which were ~10 Hz in
EpiAirway™ and 28 Hz in MucilAir™ tissues. While the reconstructed tissues presented
the advantage that detection of CBF was possible, artificial stimulation of CBF by manual
manipulation of the insert, differences due to interdonor differences or damage by long
shipping times are disadvantages compared to in-house models based on cell lines.

4.2. Alveolar Models

The suitability of A549 cells in mono and in coculture with THP-1 macrophages for
the assessment of acute and prolonged effects of particles was shown previously [27].
Assessment of the effect on TEER values is, however, only possible in the reconstructed
tissues. Similar to EpiAirway™ and MucilAir™ tissues, marked differences in TEER values
were reported for EpiAlveolar™ tissues. We obtained TEER values of 1229 ± 51 Ω × cm2
for the EpiAlveolar™ tissues with THP-1 and 1071 ± 111 Ω × cm2 for EpiAlveolar™
tissues without THP-1 at 7 d. These data are within the range of the reported values and
indicate intact barrier properties. In addition to differences in equipment and operations
for the measurements, the shipment of the tissues may impair their viabilities resulting in
decreased TEER values compared to those at the producer. TEER values at 7 d and 14 d
in the laboratory of the producer were ~1400–1000 Ω × cm2 and in another laboratory
~400–600 Ω × cm2 [17].
IL-6 levels released by EpiAlveolar™ tissues were reported to vary between 500 pg/mL
and 3000 pg/mL in the tissues with THP-1 and between 1500 and 2500 pg/mL in the tissues
without THP-1 macrophages [17]. Levels of secreted IL-8 measured over 21 days have
been reported as ~5000–15,000 pg/mL in the tissues with THP-1 macrophages and as
~20,000 pg/mL in the tissues without THP-1 macrophages. In this study, IL-6 and IL-
8 secretions were in the same order of magnitude as data published by Barosova et al.
(e.g., 214 pg/mL–1166 pg/mL in the tissues with THP-1 and 164–1944 pg/mL in those
without THP-1 for IL-6 and 5600 pg/mL–26,083 pg/mL in the tissues with THP-1 and from

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Nanomaterials 2023, 13, 682

7476–30,524 pg/mL in EpiAlveolar™ tissues without THP-1 for IL-8). Similar to that study,
we did not detect a major influence of THP-1 macrophages on the basal secretion of the
interleukins. Upon LPS stimulation, increases were in a similar range for EpiAlveolar™
tissues with and without THP-1 for IL-6 and IL-8 secretions. Coculture with THP-1, by
contrast, increased cytokine release markedly upon stimulation with LPS. Increased release
of a various proinflammatory cytokines upon LPS stimulation by A549/THP-1 cocultures
compared to A549 monocultures has been reported and was reported to be caused by
activation of NF-κB [49,50]. The lack of a pronounced response in the EpiAlveolar™ tissues
may be due to anti-inflammatory effects by the other cells present in the tissue construct.

4.3. Effects of CNT4003

All models for the bronchial tract were stable over 14 d of evaluation and reacted to the
proinflammatory stimulus LPS with significant cytokine increases. They were, therefore,
regarded as suitable to detect potential toxic effects of CNT4003. The administered dose was
2 × 25 μg CNT4003, which is in the range of the lifetime lung exposure of workers to CNTs
(12.4–46.5 μg/cm2 lung surface [51]). Exposure to CNT4003 particles caused no adverse
effects on viability, epithelial barrier integrity in bronchial models and inflammation. The
significant decrease of IL-6 levels in Calu-3 and MucilAir™ tissues might be explained
by the anti-inflammatory action of DPPC bound to the CNTs [52]. Lack of effects on cyto-
toxicity, oxidative stress generation and inflammation was also reported after cumulative
(5 × 10 μg/cm2 ) MWCNT exposure of human lung explants [19]. They observed, however,
a small increase in CBF from ~8 to ~10 Hz, which was not seen in this study. One explana-
tion may be that the high basal CBF made a further increase impossible. The model, on the
other hand, reacted to LPS exposure with a decrease in CBF, indicating that it is capable of
identifying adverse effects. We observed in this study that the distribution of the CNT4003
on the insert was not homogenous (Figure S1, Supplementary Material), and one group
reported differences in CBF between central and peripheral areas of the insert [18]. Also
due to differences in the deposition of the CNTs (Figure S1, Supplementary material) and
the fact that not the entire tissue can be evaluated, small local effects may be overlooked.
The two models for the alveolar part of the respiratory tract indicated adverse effects
only in the EpiAlveolar™ tissues with and without THP-1 macrophages by increased
IL-6 secretion upon exposure to 12.5 and 25 μg CNT4003 with no dose dependency. The
greater cytokine release induced by CNT4003 in the EpiAlveolar™ tissues than in the
A549 model is in contrast to the more pronounced response of the A549 model to LPS.
Greater toxicity of SDS in A549 than in MucilAir™ tissues has been reported and can be
explained by the lack of a mucus layer [37]. Protection of the epithelial cell by greater
surface coating may also explain the greater sensitivity of the EpiAlveolar™ tissues to
CNT4003 particles. It was reported that the surface tension, as an indication for surfactant
production, of EpiAlveolar™ tissues was markedly lower than that of A549 cells [17].
Surfactant is produced by alveolar epithelial type II cells, which represent only a small
fraction of the EpiAlveolar™ tissues, whereas the A549 cells resemble these cells and are
capable to produce surfactant [13].

5. Conclusions
Cytotoxicity, TEER values, interleukin secretion and CBF show no indication of adverse
effects of CNT4003 in the bronchial part of the respiratory tract. The increased IL-6 secretion
of the EpiAlveolar™ tissues may indicate adverse effects of CNT4003 on the alveolar part
of the lung. Since the doses for whole-life exposure of workers were applied over a shorter
time, the physiological relevance is not clear. Although the reconstructed tissues are
produced in a standardized way, considerable variations in basal parameters between
batches were seen. In addition to the interdonor differences, the duration of the delivery
from the producer to the laboratory of the user has an effect on the viability and reaction of
the tissues.

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Nanomaterials 2023, 13, 682

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/nano13040682/s1. Figures S1–S4: Distribution of CNT4003 on
cell-grown inserts; cytotoxicity of CNT4003 in Calu-3 cells; cytotoxicity of CNT4003 in A549 cells;
cytotoxicity of CNT4003 in EpiAlveolar™ tissues; Table S1: LPS-induced cytokine secretion by
EpiAlveolar™ tissues; Video S1: Mucociliary clearance in MucilAir™ tissues.
Author Contributions: Conceptualization, E.F.; methodology and data curation, C.M., R.J., M.A.-N.;
writing—original draft preparation, C.M. and E.F.; writing—review and editing, C.M., E.R. and E.F.;
visualization, M.A.-N. All authors have read and agreed to the published version of the manuscript.
Funding: PETA Science Consortium International e.V. supported this research with an Award from
MatTek Life Sciences for 3D human tissue models to assess respiratory toxicity to E.F.
Data Availability Statement: Datasets analyzed or generated during the study are available upon
request from the authors.
Conflicts of Interest: The authors declare no conflict of interest.

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induced inflammatory response in alveolar epithelial cell/macrophage co-culture. Exp. Ther. Med. 2020, 20, 76. [CrossRef]
[PubMed]
50. Grabowski, N.; Hillaireau, H.; Vergnaud-Gauduchon, J.; Nicolas, V.; Tsapis, N.; Kerdine-Römer, S.; Fattal, E. Surface-Modified
Biodegradable Nanoparticles’ Impact on Cytotoxicity and Inflammation Response on a Co-Culture of Lung Epithelial Cells and
Human-Like Macrophages. J. Biomed. Nanotechnol. 2016, 12, 135–146. [CrossRef] [PubMed]
51. Gangwal, S.; Brown, J.S.; Wang, A.; Houck, K.A.; Dix, D.J.; Kavlock, R.J.; Hubal, E.A. Informing selection of nanomaterial
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 nanomaterials

Article
The Effects of Titanium Dioxide Nanoparticles on Osteoblasts
Mineralization: A Comparison between 2D and 3D Cell
Culture Models
Gabriela de Souza Castro 1,† , Wanderson de Souza 2,† , Thais Suelen Mello Lima 2 , Danielle Cabral Bonfim 3 ,
Jacques Werckmann 4 , Braulio Soares Archanjo 5 , José Mauro Granjeiro 2 , Ana Rosa Ribeiro 6
and Sara Gemini-Piperni 1,7, *

1 Postgraduate Program in Odontology, Unigranrio, Duque de Caxias 25071-202, Brazil

2 Directory of Life Sciences Applied Metrology, National Institute of Metrology Quality and Technology,
Rio de Janeiro 25250-020, Brazil
3 LabCeR Group, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-901, Brazil
4 Visitant Professor at Brazilian Center for Research in Physics, Rio de Janeiro 22290-180, Brazil
5 Materials Metrology Division, National Institute of Quality and Technology, Rio de Janeiro 25250-020, Brazil
6 NanoSafety Group, International Iberian Nanotechnology Laboratory, 4715-330 Braga, Portugal
7 Labεn Group, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-901, Brazil
* Correspondence: [email protected]
† These authors contributed equally to this work.

Abstract: Although several studies assess the biological effects of micro and titanium dioxide nanopar-
ticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior.
Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures
display several disadvantages, such as changes in cell morphology, function, and metabolism and
fewer cell–cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex
Citation: de Souza Castro, G.; de 3D environments, to better mimic the bone microenvironment. This study aims to compare the dif-
Souza, W.; Lima, T.S.M.; Bonfim, ferentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D
D.C.; Werckmann, J.; Archanjo, B.S.; models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and
Granjeiro, J.M.; Ribeiro, A.R.; their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with
Gemini-Piperni, S. The Effects of morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was
Titanium Dioxide Nanoparticles on
evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix
Osteoblasts Mineralization: A
in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore,
Comparison between 2D and 3D Cell
our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone
Culture Models. Nanomaterials 2023,
13, 425. https://doi.org/10.3390/
mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model
nano13030425 to test the effect on bone osteointegration.

Academic Editors: Andrea Hartwig

Keywords: TiO2 NPs; titanium dental implants; 3D spheroids; osteoblasts
and Christoph Van Thriel

Received: 30 December 2022

Revised: 12 January 2023
Accepted: 16 January 2023 1. Introduction
Published: 20 January 2023 The development of nanotechnology is increasing exponentially, especially in the pro-
duction of biomaterials for dental implants [1–3]. This market moved around US$10.87 billion
in 2021 and could reach US$12.49 billion in 2022 [1,4]. In this scenario, titanium (Ti) is
the metallic material most commonly used for implant applications due to its excellent
Copyright: © 2023 by the authors.
biocompatibility and osteointegration properties [2,5]; however, the failure of dental im-
Licensee MDPI, Basel, Switzerland.
plants continues to increase [6]. Although the causes are multifactorial and often related to
This article is an open access article
distributed under the terms and
microbial colonization (biofilms), new questions have been raised about the role of corro-
conditions of the Creative Commons
sion and/or wear process in the progress of implant failure [7]. Recently, Ti-like particles
Attribution (CC BY) license (https:// (mainly titanium dioxide nanoparticles (TiO2 NPs)) have been found in the peri-implant
creativecommons.org/licenses/by/ mucosa and bone cells [6,8,9].
4.0/).

Nanomaterials 2023, 13, 425. https://doi.org/10.3390/nano13030425 https://www.mdpi.com/journal/nanomaterials

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Nanomaterials 2023, 13, 425

Prothesis degradation processes occur in all material classes, with degradation by-
products known to be hazardous. Nonetheless, nanoscale metallic debris released are of
tremendous concern since they exhibit enhanced toxicity and dissolving capacities, com-
pared with micron-size polymeric and ceramic debris [7]. Micromovements between the
implant abutment and the bone or mucosa unavoidably lead to mechanical wear of the ma-
terial (formation of nano and microsized debris), which in a corrosive environment result
in the release of metallic ions [7,10–12]. Biologically, this event is related to macrophage
activation [7,10,11,13] and the onset of pro-inflammatory response, with the release of
cytokines such as IL-1β, IL-6, and TNF-α in the peri-implant area, which culminates in re-
duced osteoblastogenesis, increased osteoclastogenesis, and bone loss in the periprosthetic
region [11,13]. Bone loss limits the ability of a prosthesis to withstand physiological loads,
giving rise to a need for revision surgery.
Many in vitro and in vivo studies have shown that titanium particles induce pro-
inflammatory and toxic effects in the peri-implant environment [7,14–17]. However, some
in vitro studies also suggest that TiO2 NPs can stimulate bone formation [2,18]. Thus,
controversy remains about the actual role of TiO2 NPs on bone cells, likely due to the
two-dimensional in vitro models used.
It has been widely reported that 2D osteoblast cell cultures [13–17,19] may lose their
original tissue organization and polarity and have limited protein–protein interactions [2,18].
They may exhibit integrins and changes in the cytoskeletal organization that alter their
original morphology [2,18,20]. In addition, cells grown in a 2D monolayer often exhibit
altered metabolism, phenotype, and gene expression, and the interactions between cells and
the extracellular matrix are different from in vivo tissues, which have a three-dimensional
architecture [2,20]. In contrast, 3D culture models exhibit greater cell–cell and cell–matrix
interactions, which are closer to the in vivo model [21]. Moreover, cellular polarity, which is
important for cellular organization and functionality, remains unaltered [2,21,22]. Therefore,
surface receptors can bind to extracellular matrix proteins, activate cellular biochemical
signals, and influence cell proliferation, differentiation, and mineralization [2,21].
Therefore, in this work, the effects of TiO2 NPs on the differentiation and production of
a mineralized matrix of human osteoblasts cultured in monolayer (2D) and osteoblast-like
spheroid culture models (3D) were investigated. Cell viability, morphology, differentiation,
mineralization, and nanoparticle internalization were investigated after exposure to NPs.
Results demonstrate that TiO2 NPs lead to a dose-dependent increase in mineralization,
although these TiO2 NPs are clinically known for their possible immune system activation.

2. Materials and Methods

Titanium anatase dispersion: TiO2 NPs (SIGMA, Kanagawa, Japan) with primary
particle size < 25 nm and surface area of 45–55 m2 /g were suspended in ultrapure water
(2 mg/mL; pH 4) and dispersed using a direct ultrasound (Q-Sonica) equipped with a
19 mm tip. The sonication was carried out in an ice bath at 32 W of acoustic delivery
power for 15 min with 8 s (pulse mode on) and 2 s (pulse mode off), following a protocol
previously described by the group [2,5]. After 24 h of stabilization, particle size and
particle agglomeration (zeta potential analysis (ζ (mV)) and the polydispersion index
(PdI)) were determined by dynamic light scattering (DLS, Zeta-Sizer Nano ZS, Malvern
Instruments GmbH, Malvern, UK). DLS measurements were performed at 25 ◦ C using
10 mm polystyrene disposable cuvettes.
To confirm particle size in the medium culture, titanium particles were alterna-
tively suspended in high glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco,
Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1 mg/mL
bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) to avoid particle re-
agglomeration.
Cell culture: The human osteoblast cell line (SAOS-2) was supplied by the Cell Bank
of Rio de Janeiro (BCRJ, Rio de Janeiro, Brazil) packed in frozen ampoules and kept
in liquid nitrogen. Cells were thawed and expanded into cell culture flasks (Corning)

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Nanomaterials 2023, 13, 425

with DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (PS—
10,000 units/mL of penicillin and 10,000 μg/mL of streptomycin) (PS, Gibco) in a humidi-
fied incubator (5% CO2 , 37 ◦ C). Cell contamination with bacteria, fungi, or mycoplasma
was analyzed as previously reported [2,5]. For the 2D model, 10,000 cells/well were seeded
in standard flat-bottom 96-well plates for 24 h.
3D culture: For 3D spheroid formation, 96-well U-bottom plates (Corning, Corning,
NY, USA) were coated with a thin layer of 1% ultrapure agarose (Sigma-Aldrich), and
10,000 cells were seeded in each well in 200 μL DMEM high glucose medium supplemented
with 10% FBS and 1% PS and then incubated for 3 days. Cell growth, shape, and mor-
phology were analyzed on an inverted optical microscope (Nikon Eclipse, Tokyo, Japan),
following a protocol previously described [2].
3D cell cytoskeleton: Spheroids were washed with 0.01 M PBS and then fixed with 4%
paraformaldehyde (PFA). The cell membrane was then permeabilized by incubation for 2 h
with 0.2% BSA + 0.1% Triton X-100 in 0.01 M PBS for 2 h at room temperature (RT). The
spheroids were washed three times with a blocking solution containing 50 nM NH4 Cl in
0.01 M PBS. Phalloidin solution (500 ng/mL, stock 1:40, ThermoFisher Lot: F432) diluted in
0.2% BSA + 0.1% Triton X-100 in 0.01 M PBS was then added and incubated for 2 h, and
later an additional 30 min with DAPI (1:500) (SIGMA-ALDRICH—Lot: 583-93-7). Cell
morphology was visualized using a confocal fluorescence microscope (DMI 6000, Leica,
Teaneck, NJ, USA).
NPs exposition: Both 2D and 3D cell cultures were exposed to 0, 5, and 100 μg/mL
TiO2 NPs suspended in incomplete osteogenic medium composed of DMEM supplemented
with 10% FBS, 50 μg/mL of ascorbic acid (Sigma), 100 Mm of β-glycerophosphate (Sigma),
and antibiotics for 3 and 21 days. Cells without TiO2 NPs treatment were used as control.
Cytotoxicity assay: After NPs exposition, the cells were washed three times with
0.01 M PBS and then incubated with 0.125% Trypsin (kept in a humidified incubator
with 5% CO2 , 37 ◦ C) for 5 min. Trypsin was blocked by adding culture medium with
10% FBS, and 2D adherent cells and 3D spheroids were mechanically dissociated. The cells
were centrifuged for 7 min at 500 x g (4 ◦ C), and the pellet was resuspended in 100 μL
annexin-binding buffer (Dead Cell Apoptosis Kit for Annexin V; Kit Life and Dead, Life
Technologies). The samples were incubated for 15 min (RT) in 3 μL annexin/fluorescein
(FITC) solution and 1 μL propidium iodide (according to the manufacturer’s instructions).
All analyses were performed in a flow cytometer (FACSAria III, BD Biosciences, Franklin
Lakes, NJ, USA).
Morphology analysis: Cells were washed with 0.01 M PBS and processed for scanning
(SEM) and transmission (TEM) electron microscopy. Briefly, cells were fixed using modified
Karnovsky (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer,
pH 7.2) for 2 h at RT and washed with 0.1 M cacodylate buffer. The samples were post-
fixed with 1% osmium tetroxide in cacodylate buffer (1:1) for 30 min in the dark, then
washed with cacodylate buffer and dehydrated in ethanol (VETEC—1567). Then, for
TEM analysis, samples were contrasted in a bloc with 1% of uranyl acetate, dehydrated
in acetone, and embedded in Spurr. Ultra-thin sections were also analyzed using EDS
in scanning transmission electron microscopy (STEM) mode in a TITAN 80–300 electron
microscope (FEI, Netherlands (300 kV).
Alternatively, for SEM analysis, the cells were dried at a critical point (Autosamdri® -
815, Series A) and metalized with gold (in a current of 40 mA for 90 sec). The 2D cell
samples were analyzed in a scanning electron microscope (JEOL Field Emission Gun-JSM-
7401F) with an acceleration voltage of 1 kV. The 3D cells were analyzed under a helium
ion beam microscope (HIM) (Carl Zeiss Orion Nanofab—beam current of 0.8 pA, using an
electron flood gun to compensate for the positive charge).
Differentiation and analysis of the cell matrix: Cell differentiation was evaluated by
alkaline phosphatase histochemistry. The cells were cultured at different times (3, 7, and
21 days). The alkaline phosphatase labeling kit (Sigma-Aldrich Lot: APF-1KT) was used,
which is based on the application of 500 μL of diazonium and naphthol salt solution for

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Nanomaterials 2023, 13, 425

30 min in the dark. Afterward, the reaction was stopped with tridistilled water. Positive
cells marked in red were photographed under an inverted optical microscope (Nikon
Eclipse TS100), using the photo program (Leica Applications Suites—LAS EZ).
To evaluate the production of the mineralized matrix, alizarin red staining was per-
formed after 3, 7, and 21 days of culture. Cells were fixed with 4% PFA and exposed
to 1% alizarin red solution (Sigma-Aldrich) at RT for 30 min, then rinsed with ultrapure
water. To quantify matrix mineralization, alizarin red-positive nodules were dissolved in a
solution of 0.5 N HCl with 5% SDS. The optical density (OD) values of absorbance were
quantified spectrophotometrically at a wavelength of 450 nm using a microplate reader
(Biotek Synergy 2 multi-mode detection with gen5 software).
Statistical analysis: Data were presented as mean ± standard deviation (SD). The
Gaussian distribution of the samples was tested, and the statistical significance of the data
was evaluated using one-way ANOVA or unpaired t-tests. The p values are shown in the
figures and statistical significance was considered when p < 0.05. Each experiment was
performed three times, with triplicates.

3. Results
3.1. Characterization of TiO2 NPs
TiO2 NPs with a primary size of 25 nm were used to mimic the wear particles released
by dental implants. The physicochemical characterization of the primary TiO2 NPs was
already published [2,5]. TEM micrographs revealed that TiO2 NPs in ultrapure water were
agglomerated, requiring the implementation of a dispersion protocol (Figure 1A). Dark-
field STEM images show the morphology and agglomeration of TiO2 NPs after dispersion
(direct probe sonication), and the STEM/EDS Ti-K map (in blue) confirmed the identity
of the TiO2 NPs (Figure 1B). DLS analysis (Figure 1C) showed that the mean diameter
(DH (nm)) of the TiO2 NPs was 135 ± 24 nm in water and increased significantly (p < 0.05,
unpaired t-test) in cell culture medium (156 ± 14 nm), maintaining a polydispersion index
(PdI) of less than 0.2. Finally, the zeta potential analysis (ζ (mV)) in water and culture
medium showed a significant decrease in the zeta potential value after medium contact,
indicating the formation of protein and ionic corona on TiO2 NPs surface (p < 0.05, unpaired
t-test) (Figure 1C).

3.2. Effect of TiO2 NPs on the Morphology of 2D and 3D Human Osteoblasts

In this study, human osteoblasts (SAOS-2) were cultured as monolayers or spheroids.
After 72 h of seeding, cells were exposed for 72 h to 100 μg/mL of TiO2 NPs. Optical
microscopy images show the conventional SAOS-2 morphology (Figure 2A, left panel).
In monolayers, the cells exhibit an epithelial-like phenotype, which is maintained after
exposure to TiO2 NPs. SAOS-2 spheroids have a round shape with a well-organized
cytoskeleton (Figure 2B,C), also maintaining their morphology upon titanium exposure
(Figure 2C). However, a 29% increase (p = 0.0151, unpaired t-test) in diameter and volume
was observed after exposition to TiO2 NPs (Figure 2D).
To confirm whether ultrastructural changes occurred after treatment with TiO2 NPs,
scanning electron microscopy (SEM) analysis was performed and showed that SAOS-2 in 2D
and spheroids (3D) maintained their morphology after 3 days of exposure to TiO2 , without
changes in their cell–cell contact (Figure 3A,B). Moreover, SEM-EDS analysis confirmed the
presence of TiO2 NPs on the surface of both cell models. A detail of interaction of TiO2 NPs
with spheroids (Ti-k, marked in blue) can be observed in Figure 3C.

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Nanomaterials 2023, 13, 425

Figure 1. Characterization of TiO2 NPs: (A) Transmission electron micrographs (TEM) of TiO2 NPs in
ultrapure water without dispersion. (B) Dark-field STEM micrographs of TiO2 NPs after dispersion
in water and cell culture medium and STEM/EDS Ti-K map confirming titanium presence (in blue).
Scale bar: 100 nm. (C): Hydrodynamic diameter (DH (nm)) and polydispersity index (PDI) of TiO2
NPs after dispersion in ultrapure water and cell culture medium obtained by dynamic light scattering
(DLS) and analysis of surface charge by zeta potential of TiO2 NPs (ζ (mV)) in water and culture
medium. The results represent the mean ± standard deviation of three independent experiments
performed in triplicate of measurement (* p < 0.05 vs. TiO2 NPs in water).

Figure 2. Treatment of TiO2 NPs in 2D and 3D cultures of human osteoblasts (SAOS-2): (A) Phase
contrast micrograph of SAOS-2 in 2D with or without 100 μg/mL of exposition to TiO2 NPs for 72 h
(scale bar: 200 μm) (B) Spheroid cytoskeleton (3D), nucleus in blue (stained with DAPI) and the actin
filaments in red (stained for F-actin) (scale bar: 100 μm). (C) Phase contrast micrograph of SAOS-2 in
3D with or without 100 μg/mL of exposition to TiO2 NPs for 72 h (scale bar: 200 μm). (D) Average
diameter and volume of spheroids. The baseline condition was used as a control. The results are
representative images of three independent experiments.

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Nanomaterials 2023, 13, 425

Figure 3. Morphology of human osteoblasts (SAOS-2) cultured in 2D and 3D after exposure to TiO2
NPs: (A) Scanning electron micrographs showing SAOS-2 cultured in 2D and in spheroids (B) after
treatment with 100 μg/mL of TiO2 NPs for 3 days. (C) STEM/EDS Ti-K map analysis. Ti showed in
blue point interacting with cells. Control: cultivation without NPs. The results are representative
images of three independent experiments. (scale bar: 40 μm, 20 μm, 50 μm and 5 μm).

3.3. Effect of TiO2 NPs on Human Osteoblast Viability

Flow cytometry analysis with PI/annexin after 3 and 21 days of culture did not show
TiO2 NPs cytotoxicity, both in the monolayer (Figure 4A) and in the spheroids models
(Figure 4B). The levels of apoptosis and necrosis were similar in all conditions evaluated.

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Nanomaterials 2023, 13, 425

Figure 4. Viability of 2D and 3D human osteoblasts (SAOS-2): SAOS-2 were exposed to TiO2 NPs
(100 μg/mL) for 3 and 21 days and the PI/Annexin assay was performed by flow cytometry of cells in
(A) 2D and (B) 3D. The baseline condition was used as a control. The results are the mean ± standard
deviation of three independent experiments (no statistical difference).

3.4. Internalization of TiO2 NPs in 2D and 3D Culture of Human Osteoblasts

Transmission electron microscopy (TEM) showed, both in 2D and 3D models, the inter-
nalization of TiO2 NPs, that preferentially located in the cell cytoplasm within membrane-
like-vesicles or after cell-membrane disruption, possibly in multivesicular bodies (MVBs)
or auto-phagolysosomes delimitated by the membrane (Figure 5).

3.5. Differentiation and Mineralization of Human Osteoblasts after Exposure to TiO2 NPs
To understand the influence of TiO2 NPs on the differentiation and mineralization
of both cell models (2D and 3D), analyses of alkaline phosphatase (ALP) (differentiation
marker) and alizarin red (mineralization marker) were performed. For these analyses,
osteoblasts were cultured for up to 14 days, and two exposure concentrations (5 and
100 μg/mL) of TiO2 NPs were used. Previous data in human primary osteoblasts 2D
histochemical micrographs showed that the treatment of TiO2 NPs did not enhance the
labeling for ALP (marked in red) after 14 days of culture (Figure S1A). However, in the
mineralization analysis, there was a dose-dependent increase in alizarin staining after
14 days of treatment with 100 μg/mL (marked in intense red) compared to the control
(Figure S1B).
To compare differences in mineralization occurring in 2D and 3D models, we per-
formed alizarin staining after 3, 7, and 14 days after 5 μg/mL or 100 μg/mL TiO2 NPs
exposure in both models. Alizarin red results showed a significant dose-dependent in-
crease in mineralization at 14 days compared to the control, both in 2D (Figure 6A) and
3D (Figure 6B). Moreover, when treatment values are normalized by control values, the
mineralization increase is higher in the 3D model when compared with the 2D model,
suggesting that both models can present different results in the mineralization evaluation
(Figure 6C and representative images in Figure 6D).

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Nanomaterials 2023, 13, 425

Figure 5. Internalization of TiO2 NPs: Transmission electron microscopy (TEM) micrographs showing
TiO2 NPs internalization in human osteoblasts (SAOS-2) 72 h cultured: (A) control, cultured without
NPs or (B,C) treated with 100 μg/mL of TiO2 NPs (black arrow). The results are representative
images of three independent experiments. (scale bar: 1 μm, 2 μm, and 200 nm).

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Nanomaterials 2023, 13, 425

Figure 6. Mineralized matrix produced by human osteoblasts (SAOS-2) in 2D and 3D cells exposed
to TiO2 NPs (0, 5 and 100 μg/mL) during 3 and 14 days in osteogenic medium. Alizarin red
assay shows a dose-dependent matrix production in (A) 2D SAOS-2 model and (B) 3D SAOS-2
models. The graphics in (C) present a comparison between the inorganic matrix production on both
2D and 3D models at 14 days (final experimental time) normalized vs each control to show dose-
dependent mineralization fold increase in the different models. The results are average ± standard
deviation of three independent experiments * p < 0.05; (D) representative images obtained through
optical microscopy.

4. Discussion
Titanium is the main material employed in the dental implant industry, due to its high
mechanical strength, low elastic modulus, corrosion resistance, ductility, and biocompatibil-
ity [6,9]. However, tribocorrosion processes at the implant surface lead to accelerated bone
loss, compromising osseointegration, and increasing periprosthetic failure [2,5–9,23,24].
The hostile electrolytic environment (oxidation/reduction) together with mechanical action
at the interface enables the tribocorrosion phenomena [7,10]. As a consequence, degradation
products (released from implants) including metal ions, micrometric, and/or nanomet-
ric metallic debris (TiO2 NPs) can be internalized by cells in the bone niche, possibly
generating cytotoxic effects [6,9,10]. The adverse effects of TiO2 NPs vary widely in the
literature, which raises concern among authorities and physicians due to their high preva-
lence [5,10,17]. Literature data reveal that inflammatory stimuli associated with cytokine
overproduction and increased production of reactive oxygen species are referred to as
primary toxic effects that lead to cell death [6,9,13,17].
Some authors explained that this mechanism leads to activation of immunological
sentinels and accumulation of antigens such as ions, nanoparticles, microparticles, and
bacterial antigens via the functional interface between dental implant and tissue. This leads
to immunological cell polarization and follows dental implant loss [6,9].
Most available studies that evaluate osteoblast response to TiO2 NPs use 2D cell culture
models, which have shown limitations regarding cell growth and cell–cell and cell–matrix
interactions, among others [25–28]. Few studies evaluate the influence of TiO2 NPs on the

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Nanomaterials 2023, 13, 425

physiology of bone cells grown in 3D models such as spheroids [2]. Osteoblast spheroids
can be considered as a culture model that better mimics living cells in terms of structural and
biofunctional properties and provides more reliable results compared to conventional 2D
cell cultures (Figure 7) [2,25,29]. Despite this, there are some limitations to spheroid culture,
mainly because cellular environments are not similarly exposed to the culture medium.
This can lead to the formation of a microenvironment inside the spheroids that can select
groups of cells [30,31]. Partial diffusion of nutrients or oxygen can induce necrotic areas in
the central area of the spheroids [32]. However, well-characterized multicellular spheroids
exhibit different levels of extracellular matrix deposition, growth factor secretion, and gene
expression profiles [2]. The viability, morphology, and gene expression of osteoblastic
spheroids are contact-dependent, and single or co-culture spheroids have been shown to
have an impact on bone cell function [33]. Interestingly, a study reported that primary
osteoblasts and pre-osteoblasts MC3T3-E1 can differentiate into osteocytes when grown in
3D cultures [34]. Therefore, 3D culture models can be used to study the pathophysiological
reactions of TiO2 NPs in bone metabolism compared to 2D cultures. A previous study by W.
Souza et al., on the cytotoxicity effect of TiO2 NPs on osteoblast spheroids, revealed that 72 h
exposition to TiO2 NPs can alter the cell cycle, without interfering with osteoblasts’ ability
to differentiate and mineralize and significantly increase collagen and pro-inflammatory
cytokine secretion [2]. In the present study, a longer exposure period (21 days) was
assessed to compare 2D with 3D osteoblasts models to better understand their relevance
for nanotoxicological studies.

Figure 7. Scheme of differentiation and production of a mineralized matrix of human osteoblasts

SAOS-2 cultured in monolayer and 3D spheroid cell models after exposition to TiO2 . After high
TiO2 NPs exposure, the dose-dependent increase of bone-mineralized matrix in the 3D cells model is
higher than in monolayer (2D) culture.

TiO2 NPs are chemically stable, have antibacterial properties, and induce less toxicity
than other nanostructures, and, when exposed to the biological environment, blood plasma
proteins and ions selectively adsorb on the outer surface of the cell [35]. The complex
interface depends on the physical and chemical characteristics of the NPs, as well as the
biological characteristics of the environment [36]. In the present study, we observed that
TiO2 NPs had an average size of 150 nm in the culture medium. We can notice an increase
in the average size after the addition of the culture medium due to the adsorption of
proteins and ions on the TiO2 NPs surface, which can be correlated with the change in
surface charge, identified by zeta potential analysis. Furthermore, in our previous study,

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we confirmed the adsorption of calcium and phosphate on the surface of TiO2 NPs, which
are important mediators of bone mineralization [5].
To understand the influence of TiO2 NPs on bone cell mineralization, we used a
mature osteoblast line, cultured both in monolayer (2D) and spheroids (3D), the former
characterized previously [2]. Spheroids and monolayer cells were treated with 100 μg/mL
of NPs for 21 days. In both models (2D and 3D), we observed the internalization of TiO2 NPs
in membrane vesicles (with 72 h). Some studies have shown that NPs can be internalized
in a dose-dependent manner, accumulating preferentially in the perinuclear region, and
having as their final destination the lysosomes [35,37]. Normally TiO2 NPs are not observed
dispersed in cell cytoplasm [5,35,37]. However, the effect of TiO2 NPs on cells is directly
related to their size distribution, crystal structure, as well as corona formation [35]. Recently,
the formation of a bio-camouflage rich in calcium, phosphorus, and hydroxyapatite crystals
around TiO2 NPs was demonstrated, which is known to facilitate the internalization in 2D
and 3D osteoblastic models since the detected chemical elements are essential for bone cell
metabolism and mineralization [2,5,38].
The present study demonstrated that TiO2 NPs did not alter the viability of osteoblasts
in both cell models (with 21 days). Concomitantly, they did not change the osteoblast
morphology or spherical shape of the 3D model upon internalization of the NPs. Interest-
ingly, they were able to stimulate an increase in calcium deposition, which is indicative of
the activation of a mineralization process in osteoblastic spheroids. In the present study,
results of alkaline phosphatase synthesis and calcium labeling demonstrated that TiO2 NPs
increased osteoblast differentiation that induced greater mineralization in a 3D culture
model, suggesting that the 3D architecture possibly increases cell surface interaction with
previously reported TiO2 NPs bio-camouflaged [2]. The mineralization increase in 3D mod-
els after exposure to NPs may be related to the greater cell surface capable of contacting
NPs when compared to the monolayer (2D), enhancing the stimulatory effects of TiO2
NPs [39]. This is consistent with previous studies that reveal that 3D osteoblasts models
when exposed to TiO2 NPs, compared to monolayer cells, induce the secretion of vascular
endothelial growth factor (VEGF), activating a cascade of events resulting in higher type I
collagen production [39–43]. Bone mineralization is the first step for implant osseointegra-
tion and begins when collagen I acts as a three-dimensional scaffold for hydroxyapatite
deposition [44]. Another study reported greater osteogenic differentiation when using 3D
collagen gel culture [34]. Studies also showed that the 2D cell model does not yet seem to
be the better model to study interaction with NPs; instead, the spheroids are also promising
for application to 3D bioprinting tissue models with biomaterial scaffolds, as an innovative
technology to improve bone osteointegration [45].
Unfortunately, there is no consensus in the literature on how to evaluate the biological
effect of TiO2 NPs. Without standardized protocols to assess the biological impacts of NPs,
it is necessary to validate safe assessments and mitigate potential health impacts, moving
toward the evaluation and development of new cellular study models to better mimic
the biological environment [37]. Although osteoblastic spheroids have their advantages
compared to monolayers—such as reproducibility, better nutrients, oxygen diffusion gra-
dients, improved cell–cell interactions, matrix deposition, and models with various cell
stages (proliferating, quiescent, apoptotic, hypoxic, and necrotic cells) [46,47], 3D spheroid
models have not been validated as realistic in vitro models [29,46,48]. One of the main
drawbacks of spheroids is that the porosity and mechanical properties is difficult to be
studied. Thus, efforts should be made to improve 3D bone cell models to recapitulate the
bone microenvironment that is known to be constituted by different cell types and has
dynamic and metabolic activity.
TiO2 NPs released from dental implants are, on the one hand, considered the cause
of clinical peri-implant bone loss; on the other hand, they may be able to stimulate the
production of a mineralized extracellular matrix in osteoblast spheroids [48]. Another
important aspect is that spheroids can respond physiologically better to the stimuli of TiO2
NPs, which corroborates the development of new studies to create new models applied

33
Nanomaterials 2023, 13, 425

in clinical studies, to favor the process of bone remodeling and alternative treatment for
periodontitis and peri-implantitis. In addition, the spheroids themselves can be applied to
high-cell-density tissue models, innovative technology for bone augmentation, and soft
tissue replacement procedures [45]. Therefore, the combination of TiO2 NPs with spheroid
cells should be an interesting approach for tissue reconstruction.
Lastly, our results demonstrate that TiO2 NPs increase calcium deposition in 3D versus
2D cultures. Although this study revealed interesting findings regarding the behavior and
role of TiO2 NPs in generating stimuli for mineralization in 3D models only, it should be
noticed that our results are limited to the conditions tested and the experimental setup.
Further studies should be encouraged, and further evaluations using the quantification of
genes that act on differentiation and mineralization should be performed. However, our
results help to better understand the possible impact of 3D culture in dentistry, and also
open a discussion about the dual role of TiO2 NPs, which on one side can activate an inflam-
matory response that leads to bone resorption. However, on the other hand, it is activating
mineralization. Our findings are considered clinically relevant, since, for the first time, we
report that at the bone-implant interface, TiO2 NPs besides the activation of macrophages
can also stimulate osteoblasts that play a fundamental role in the mineralization process.

5. Conclusions
In this study, the cells were exposed to TiO2 NPs at concentrations up to 100 μg/mL
in 2D and 3D models for up to 21 days of exposure.
TiO2 aggregates were dispersed to nanometric size and characterized successfully. Its
internalization in both cell models showed no differences in cell morphology or viability
and bone mineralization induction in a dose-dependent form in both culture models.
However, the mineralization process was more intense in the 3D spheroid culture
compared to the 2D monolayer model.
This brings a new discussion about the possible advantages of TiO2 NPs on bone
mineralization, which may suggest that the action of nanometric particles can contribute to
the osseointegration process in titanium dental implants, reducing periprosthetic failures
and using 3D cell models as an innovative technology to improve bone osteointegration
induced by nanoparticles.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/nano13030425/s1, Figure S1. Primary osteoblast differentiation and
mineralization: alkaline phosphatase (A) and alizarin red (B) staining in primary human osteoblasts
exposed during 3, 7, and 14 days after exposure to 5 μg/mL or 100 μg/mL TiO2 NPs. The results are
representative images of three independent experiments. (scale bar: 200 μm).
Author Contributions: S.G.-P., A.R.R., W.d.S. and G.d.S.C. designed the study. G.d.S.C., W.d.S.,
S.G.-P., A.R.R., T.S.M.L. and J.W., performed experimental work and analyses. G.d.S.C., W.d.S.,
D.C.B., S.G.-P., A.R.R., J.W., J.M.G. and B.S.A. contributed technical support and discussion. G.d.S.C.,
W.d.S., S.G.-P., A.R.R., T.S.M.L., D.C.B. and J.M.G. wrote/edited the manuscript. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was supported by National Council for Scientific and Technological Development
(CNPq) with grants 405030/2015-0, 306672/2016-2, and 467513/2014-7. J.M.G. thanks the Cientista do
Nosso Estado award from FAPERJ and CNPq/Faperj—National Institute of Science and Technology
in Regenerative Medicine (INCT-Regenera, process n. 465656/2014-5). A.R.R. thanks the Sinfonia
project H2020 of the European Union (N.857253).
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.

34
Nanomaterials 2023, 13, 425

Acknowledgments: We thank the Physical Research Centre (CBPF), the National Institute of Metrol-
ogy, Quality and Technology (INMETRO), and the Federal University of Rio de Janeiro (UFRJ),
especially Maria Isabel Rossi, for all the support given to the biological experiments. The authors
thank Suzana Azevedo dos Anjos for her valuable help in carrying out the sample preparation for
SEM, and Mariana Moreira for her help in obtaining micrographs in SEM and TEM. The authors
thank the Cell Bank of Rio de Janeiro (BCRJ), especially Radovan Borojevic.
Conflicts of Interest: The authors declare no conflict of interest.

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 nanomaterials

Article
Different Sensitivity of Advanced Bronchial and Alveolar
Mono- and Coculture Models for Hazard Assessment
of Nanomaterials
Elisabeth Elje 1,2, *, Espen Mariussen 1,3 , Erin McFadden 1 , Maria Dusinska 1 and Elise Rundén-Pran 1, *

1 Health Effects Laboratory, Department for Environmental Chemistry, NILU—Norwegian Institute for Air
Research, 2007 Kjeller, Norway
2 Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0372 Oslo, Norway
3 Department of Air Quality and Noise, Norwegian Institute of Public Health, 0456 Oslo, Norway
* Correspondence: [email protected] (E.E.); [email protected] (E.R.-P.); Tel.: +47-63-89-81-98 (E.E.)

Abstract: For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new
approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R’s
to reduce, replace and refine animal experiments. This study aimed to establish and characterize
an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated
at the air–liquid interface (ALI), both as monocultures and in cocultures with human endothelial
EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar
model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were
exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability
(alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage
(enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay)
were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the
cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell
viability and DNA strand breaks. This study indicates that the four ALI lung models have different
Citation: Elje, E.; Mariussen, E.; sensitivities to NM-300K exposure and brings important knowledge for the further development
McFadden, E.; Dusinska, M.; of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based
Rundén-Pran, E. Different Sensitivity on NAMs.
of Advanced Bronchial and Alveolar
Mono- and Coculture Models for Keywords: NAMs—new approach methodologies; ALI—air–liquid interface; genotoxicity; BEAS-2B;
Hazard Assessment of
A549; NM-300K; DNA damage; chromosomal damage; cytokines
Nanomaterials. Nanomaterials 2023,
13, 407. https://doi.org/10.3390/
nano13030407

Academic Editors: Saura Sahu and 1. Introduction

Jean-Marie Nedelec
The production and usage of nanomaterials (NMs) are rising, increasing the risk of
Received: 25 November 2022 human exposure. Inhalation is the most important exposure route for airborne nanoma-
Revised: 3 January 2023 terials (NMs) and particulate matter (PM) in humans, making the respiratory system a
Accepted: 13 January 2023 first-target organ [1]. The respiratory tract consists of the tracheobronchial region leading
Published: 19 January 2023 into the alveolar region, where gas exchange with blood occurs across the thin lung–blood
barrier (0.4 μm) [1,2]. Besides gas exchange, a main function of the lower respiratory tract
is defense against inhaled toxicants [1]. Interaction with and deposition of inhaled NMs
are likely to occur in the bronchial and alveolar region. Particle deposition is dependent
Copyright: © 2023 by the authors. upon the NMs’ physicochemical properties, such as size and solubility [2].
Licensee MDPI, Basel, Switzerland.
NMs and their dissolved compounds can cause primary effects in the respiratory
This article is an open access article
system, or secondary circulatory effects after crossing the lung–blood barrier and taken
distributed under the terms and
up in the blood. A human study has shown the translocation of inhaled gold NM or
conditions of the Creative Commons
its dissolved species into the circulatory system and accumulation at sites of vascular
Attribution (CC BY) license (https://
disease [3]. Gold was detected in blood and urine up to three months after inhalation
creativecommons.org/licenses/by/
4.0/).

Nanomaterials 2023, 13, 407. https://doi.org/10.3390/nano13030407 https://www.mdpi.com/journal/nanomaterials

39
Nanomaterials 2023, 13, 407

exposure to gold NMs [3,4]. Translocation of silver NMs has been seen in in vivo studies in
rodents [5].
In order to comply with the 3R´s principle to reduce, refine and replace animal
experiments, new advanced in vitro models are developed to better simulate the complexity
of human lungs. Reliable in vitro models of the airway system are of critical importance
for the risk assessment and governance of NMs and other environmental pollutants [6,7].
Human cells cultured on a microporous membrane at the air–liquid interface (ALI) with cell
culture medium only at the basolateral side, represent a highly relevant model for inhalation
toxicity studies [8]. Human lung cell lines such as A549 and BEAS-2B are commonly used as
model cells in respiratory toxicology. A549 cells are alveolar type-II carcinoma cells, while
BEAS-2B cells are immortalized cells from normal human bronchial epithelia. Both A549
and BEAS-2B cells form monolayers when cultivated at the ALI [9,10]. In order to further
advance the models, cocultures with other cell types, such as macrophages, dendritic cells,
or endothelial cells, can be established. The ALI exposure model aims to better mimic the
physiology of the respiratory system and is regarded as a more relevant in vitro model
compared to submerged exposure. Aerosolized exposure to the particles on top of the cells
introduces less changes in the physicochemical properties of the test substance compared
with submerged exposure [8].
Inhalation exposure to NMs, PM or other compounds may lead to adverse human
effects. Genotoxicity is a critical endpoint in the hazard assessment of chemicals, including
NMs, and should be assessed both at the level of DNA/genes and chromosomes. The
comet assay is a widely used assay for determining DNA damage as DNA strand breaks
(SBs), and as oxidized or alkylated bases by the inclusion of a repair enzyme such as
formamidopyrimidine DNA glycosylase (Fpg) [11]. For the detection of chromosomal
damage, the most-used test is the micronucleus assay (OECD test guideline 487), which
detects the formation of micronuclei from chromosomes, chromatid fragments or whole
chromosomes that lag behind in cell division [12,13]. So far, a very limited number of
studies have addressed several genotoxicity endpoints in ALI models. Our approach,
combining advanced and more physiologically relevant in vitro respiratory models and
exposure systems with genotoxicity testing (by both comet and micronucleus assays), will
support the hazard characterization of NMs for risk assessment and safe use.
NMs can induce DNA damage by direct contact with DNA, or indirectly via NM-
induced oxidative stress or intermediate molecules and processes in cells (primary genotox-
icity). Secondary genotoxicity can be driven by an inflammatory response [14]. The airway
epithelium is an integrated part of the inflammatory defense response after inhalation ex-
posure to toxicants. Pro-inflammatory cytokines are considered biomarkers of NM-induced
toxicity and can be linked with adverse effects. The pro-inflammatory cytokines IL-6 and
IL-8 are among the cytokines predominately secreted by monocytes, and both are coupled
to lung injury and considered biomarkers of lung disease [15–17]. IL-8 can also act as a
chemokine [17]. The bronchial epithelium serves as a first-line defense system against
inhaled pathogens mainly by the release of chemokines, such as IL-8 [18]. The cytokines
IL-6 and IL-8 have been shown to be secreted by airway epithelial, including BEAS-2B cells,
and endothelial cells and be involved in lung inflammation responses [18–21]. IL-6 has
been shown to be released from BEAS-2B cells after exposure to particulate matter below 1
μm in size (PM1), and both IL-6 and IL-8 were induced in BEAS-2B cells after exposure to
the PM2.5 fraction [22,23]. Endothelial EA.hy926 cells were shown to release IL-6 and IL-8
after exposure to silica NMs [19].
The A549 cell line has frequently been used in coculture lung models and has been
shown to be useful in a range of applications for hazard assessment of NMs [24–35].
The non-cancerous origin of BEAS-2B cells may make the cell line more relevant for use
in risk governance of NMs, particularly as a bronchial respiratory model. Coculture
models with BEAS-2B in ALI conditions for hazard assessment are, however, much less
characterized than those with A549. The main aim of this study was to characterize
an advanced respiratory model with BEAS-2B bronchial cells cultivated in ALI models,

40
Nanomaterials 2023, 13, 407

after exposure to an aerosolized reference silver NM, NM-300K. The cells were cultivated
both as monocultures and in cocultures with human endothelial EA.hy926 cells. Cells
from ALI cultures were analyzed for cytokine secretion, cytotoxicity, barrier integrity,
DNA damage by the comet assay and chromosomal damage by the cytokinesis-block
micronucleus assay. Importantly, the responses obtained with the bronchial BEAS-2B model
were compared with the A549 alveolar model. The experimental design brilliantly allows
for the comprehensive analysis of several endpoints from the same sample, facilitating
increased throughput, better comparability, reduced costs, and sustainability by design to
support the development of new approach methodologies (NAMs) and next-generation
risk assessment (NGRA) of NMs.

2. Materials and Methods

2.1. Experimental Design
An experimental design combining the analysis of several endpoints from the same
sample was developed. The same inserts with cells at the ALI were used for the analysis of
cytokine secretion in basolateral media (enzyme-linked immunosorbent assay, ELISA), Ag
permeation (inductively coupled plasma mass spectrometry, ICP-MS), cell viability (alamar-
Blue assay), cell proliferation, DNA damage and oxidized base lesions (enzyme modified
version of the comet assay), and chromosomal damage (micronucleus assay) (Figure 1).
In parallel, additional experiments on ALI cultures were included to further characterize
the models, and experiments with traditional submerged cultures were performed for
comparisons. For each exposure condition, 1–2 culture inserts were included from both
mono- and cocultures, and at least 3 independent experiments were performed, in order to
allow for appropriate biological variation to be included in the results and analysis.

Figure 1. Experimental design for the four different respiratory models exposed at the air–liquid
interface (ALI) in the VITROCELL® Cloud system. Created with BioRender.com.

41
Nanomaterials 2023, 13, 407

2.2. Nanomaterials
The Ag NM NM-300K is listed on the representative manufactured NMs list of the
European Commission Joint Research Centre (JRC, Brussels, Belgium) and was selected
for this study based on its toxicity in our previous work [36–39]. NM-300K was provided
by the Fraunhofer Institute for Molecular Biology and Applied Ecology (Schmallenberg,
Germany). NM-300K is a silver colloidal dispersion with a nominal silver content of 10%
w/w. The NMs were dispersed in an aqueous solution with stabilizing agents, consisting
of 4% w/w each of polyoxyethylene glycerol trioleate and polyoxyethylene (20) sorbitan
mono-laurate (Tween 20). The pristine diameter of NM-300K is about 15 nm, and the size
distribution is narrow, where >99% of particles (by number) have a size below 20 nm. A
second peak of smaller NMs of about 5 nm has also been reported. The majority of the
NMs have a spherical shape [40].
Dispersed NMs were received in vials of approximately 2.0 g each, sealed under argon.
The vials were stored at room temperature (RT) in the dark before use. The dispersion
medium, NM-300K DIS, contained the aqueous solution with stabilizing agents at the same
concentrations as NM-300K, but without Ag. This was used as a solvent control.

2.3. Nanomaterial Dispersion and Characterization

Stock dispersions of NM-300K were prepared in accordance with the Nanogenotox
protocol [41]. The original vial of NM-300K was vortexed (>10 s), before approximately 1 g
was added to a scintillation vial (Wheaton Industries, Millville, NJ, USA). To this, water
with 0.05% bovine serum albumin (BSA) was added to yield a final nominal concentration
of 10 mg/mL Ag-NMs, in order to obtain a high enough concentration of Ag-NMs in
the ALI exposure system. The total Ag and dissolved Ag species (<3 kDa fraction) were
measured from the same samples in Camassa and Elje et al. (2022), revealing a silver
concentration of 7.2 ± 0.9 mg/mL with 3.6 ± 0.1% dissolved silver species [39].
The dispersion was sonicated in an ice bath using a calibrated Q500 sonicator with a
6 mm microtip probe (Qsonica L.L.C, Newtown, CT, USA), with amplitudes of 30–40% for 7–
13 min. The energy output of the sample was 1030–1285 J/mL dispersion (n = 10), similarly
to what is recommended by the Nanogenotox protocol (1176 J/mL [41]). Additional stock
dispersions were sonicated using lower energy (95–720 J/mL, n = 5), and were included in
the study as similar results were seen compared with the other dispersions. The NM stock
dispersions were kept on ice for 10 min before use, to let the NMs settle. Before use, the
vial was vortexed for approximately 10 s. The dispersion was kept on ice throughout the
experiment. The dispersion medium NM-300K DIS (without Ag) was prepared following
the same protocol as that for NM-300K.
The NMs were previously tested for endotoxins, with endotoxin contents below the
limit of detection [39]. Stock dispersions for use in the submerged exposure experiments
were diluted to 2.56 mg/mL in BSA-water before further dilution in culture medium
(Section 2.4) in order to ensure consistency with other studies on the same NM.

2.4. Physicochemical Characterization of Nanomaterials in Dispersion

NM-300K was subjected to measurement of hydrodynamic diameter and zeta potential
in a Zetasizer Ultra Red (Malvern Panalytical Ltd., Malvern, United Kingdom) immediately
after preparation and after 24 h. The hydrodynamic diameter was determined using
dynamic light scattering (DLS) by the particles in suspension. The measured particle size
is the diameter of a sphere that diffuses at the same speed as the particle being measured,
which is determined by measuring the Brownian motion of the particles by DLS and then
interpreting the size using the Stokes–Einstein equation.
The NM stock dispersion was vortexed and diluted 1:100 in sterile filtered MilliQ
water, and a 1 mL dispersion was transferred to a disposable cuvette (DTS0012) for size
analysis. The hydrodynamic diameter was measured by non-invasive back scatter at 174.7◦
with 3–5 steps. Analysis was performed at 25 ◦ C with 120 s equilibration time, automatic
attenuation, and no pause between steps. Data were processed in the ZS Explorer software

42
Nanomaterials 2023, 13, 407

(version 2.0.0.98, Malvern Panalytical Ltd.), using general purpose model, refractive index
1.59 and absorption 0.01.
Measurement of size distribution of NMs diluted in culture medium was performed
directly after preparation and after 24 h incubation at 37 ◦ C, 5% CO2 . First, the stock
dispersion was vortexed, and mixed with serum-free LHC-9 medium (article no. 12680013,
ThermoFisher Scientific, Waltham, MA, USA) to give the highest tested concentration
(141 μg/mL or 100 μg/cm2 in submerged exposure). Then, the sample was diluted 1:10
in sterile filtered MilliQ water, transferred to a disposable cuvette and measured as de-
scribed above.
Results were presented as Z-average (Z-ave), which is the intensity-weighted mean
hydrodynamic size of the ensemble collection of particles, the polydispersity index (PDI),
and hydrodynamic diameter (by intensity) of individual peaks in the size distributions.
For zeta potential analysis, the NM stock dispersion was vortexed and diluted 1:100 in
sterile filtered MilliQ water, and 1 mL dispersion was transferred to a pre-wetted disposable
folded capillary cell (DTS1070). The zeta potential was measured at 25 ◦ C using mixed-
mode measurement phase analysis light scattering (M3-PALS).

2.5. Cell Culture

BEAS-2B cells, an Ad12-SV40 hybrid virus-transformed human bronchial epithelial
cell line [42,43], were purchased from ATCC (Manassas, VA, USA) (SV40 immortalized,
CRL-9609, LN: 62853911). The cells were cultured in serum-free LHC-9 medium without
supplements, and they were maintained in an incubator with humidified atmosphere at
5% CO2 and at 37 ◦ C. The cells were passaged two times a week at 80–85% confluency. To
facilitate detachment, the cells were incubated with trypsin-EDTA (0.25%, Sigma-Aldrich,
Saint-Louis, MO, USA) with polyvinylpyrrolidone (PVP, 0.5% wt/vol) for 3–5 min. Medium
was added, and the suspension was centrifuged to remove the trypsin/PVP before cells
were seeded at 1.3 × 104 cells/cm2 in Corning CellBind® cell culture flasks (Corning,
Corning, NY, USA). The cells were used at passages (P) 3–14 (details in Table S1).
The human alveolar type II lung epithelial A549 cells [44] were provided by ATCC,
and they were cultured in Dulbecco’s Modified Eagle’s Medium, DMEM, with low glucose
(D6046, Sigma-Aldrich) supplemented with 9% v/v fetal bovine serum, FBS (prod.no.
26140079, ThermoFisher Scientific), and 1% v/v penicillin–streptomycin (100 U/mL pen
and 100 μg/mL strep) (catalog no. 15070063, ThermoFisher Scientific). Human endothelial
EA.hy926 cells [45] were provided from ATCC and were cultured in DMEM with high
glucose (catalog no. 11960, ThermoFisher Scientific), supplemented with 9% v/v FBS,
1% v/v penicillin-streptomycin, sodium pyruvate (1 mM) and L glutamine (4 mM). The
cells were maintained in an incubator with a humidified atmosphere at 5% CO2 and at
37 ◦ C. The cell lines were passaged two or three times a week at 85–90% confluency, using
phosphate-buffered saline (PBS, catalog no. 14190094, ThermoFisher Scientific) for washing
and semi-dry trypsinization using trypsin-EDTA (0.25%) incubation at 37 ◦ C for 3 min. The
cells were seeded at 1.3 × 104 cells/cm2 in standard cell culture flasks. A549 cells were
used at P2–15 and EA.hy926 cells were used at P3–19 (details in Table S1). All cell lines
were regularly tested for mycoplasma contamination and found negative.

2.6. Cell Cultures at the Air–Liquid Interface

The seeding of mono- and cocultures were performed in a similar manner as previously
described [46,47] with some modifications. All cell types, epithelial A549 (P3–15) and BEAS-
2B (P3–14), and endothelial EA.hy926 (P3–16) (details on p numbers in Table S1), were
seeded at a density of 1.1 × 105 /cm2 . Mono- and cocultures were cultivated on permeable
cell culture inserts in 6-well plates with a porous membrane of polyethylene terephthalate
(PET) with a 1 μm pore diameter. Two insert types were used, with similar properties and
cell attachment results: Millicell (catalog no. MCRP06H48, Sigma-Aldrich) or ThinCert™
(catalog no. 392-0128, Greiner Bio-One, Kremsmünster, Austria). The same insert type was
used for all samples within an experiment.

43
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että tästä päästä saatettiin pistää sisään vihkimäsormuksia y.m., joita
tahdottiin siten vihkiä ja tehdä onnea tuottaviksi.

Oikeanpuolisessa (kuva 12) kuvataan edellä kerrottua tapausta

Länsi-Göötainmaalla, kun Pyhä Henrik rankaisee yhtä pilkkaajaansa.

Vasemmanpuolinen taulu (kuva 13) osoittaa kuinka Pyhä Henrik

auttaa jumalisia kokemäkeläisiä merimiehiä. Jo on masto katkennut
ja aallot ovat painaneet perän veden pinnan alle. Hädissään miehet
huusivat Pyhää Henrikkiä apuun, joka kohta ilmestyikin pilvissä ja
asetti myrskyn, niin että miehet, niinkuin kuvan oikeanpuolinen
puolisko näyttää, tyynellä merellä, täydessä turvassa saavat jatkaa
kulkuansa.

(J. Krohn: Suomen Kuvalehti.)

MUINAISUUDEN MUSTAN YÖSSÄ.

Muinaisuuden mustan yössä, Pohjolan ajan alussa, aaltosi

ulappa aava yli suuren Suomenmaan. Suolaisten sumujen
vyössä vyöryi synkät aallot vaan sylitellen, sylkytellen, halki
aikojen halaten jäitä hyisten huippuvuorten, päitä lauttojen
lumisten.

Aallot kulki tuulten teitä, aurinko ajan latua, hiljaa hiihti

päivät pitkät, hiljaa hiipi pitkät yöt — päivä kutoi kuiden työt,
yössä vuossadat samosi.

Vaan kun kerran päivän kaari taasen taivolle kohosi, maa oli
merestä noussut, veestä manner maatunnunna, ulapalle outo
saari, aalloille nimetön niemi.

———

Kerran illalla Kalevan kansa saapui niemehen nimettömään,

matkan vaikean vaivoistansa yöpyi rannalle lepäjämään,
sytytti savunsa saarelmalle, tulensa niemyen nenähän toi,
levitti lehviä kuusten alle, louhten lomahan majansa loi.
 Vaan kun aamulla päivyt koitti valaen vesille kultiaan,
kummun kuusessa peippo soitti, kukahti käkönen oksallaan;
silloinpa heraksi heltyi mielet, sulipa hymyhyn partasuut,
silloin helkähti sydänten kielet, humahti rinnassa "isien puut".

Juuri kaivaten maata moista lähteneet oli kulkemaan tuolta

he Aasian alkukoista, otava, aurinko oppainaan — maata
kaivaten, kussa senki luonto luontoa omaa ois, kussa Kalevan
heimon henki korven honkien humussa sois.

Kohta tietäjä lyylin laittoi, saneli pyhäksi salmen suun,

kummun kuusesta oksat taittoi, julisti vuorella Jumalan puun,
lepikön aitasi Hiiden haaksi, asuma-ahoksi vainajain, nimitti
niemyen Suomenmaaksi Ukolle uhreja teurastain.

Näinpä riemulla Kalevan kansa tuhanten järvien maahan

jäi, aneli onnea Ahdiltansa, palui palkkoja metsältäi, saarten
salmihin verkot heitti, syvälle korpehen vipunsa vei, paaden
kourussa kalansa keitti, tuntenut kaupin kaluja ei.

Vaan kun iltasin kumpuloilla päivän laskua katseltiin, tai kun

erässä nuotioilla otavan sarvia outeltiin, — silloinpa helisi
kannelkielet, kautta kumpujen soitot soi, silloin sankarit
vapaamielet sormet sormien lomahan loi,

silloin loihtuja, laulelmoita partahuulilta humisi vaan,

alkusyntyjä syviä noita, peruja kansojen kehtomaan: Vipusen
virsiä, satuja sammon, "lovessa"-käyntejä loihtijain, kuvia
kummia Kalman kammon, ituja Tuonelan tutkelmain.

Luonnon lapsena kasvoi kansa, nukkui honkien huountaan,

uneksi uudesta onnestansa, heräsi aaltojen loiskinaan; huhtia
raaten, kaskia kaaten voimoa elämän nisistä joi, metsän
Mielikin povella maaten luonnon luottehet kuulla voi.

Siellä suojana hongan juuri, toivon tähtöset päällä pään,

kasvoi Suomen sukuni suuri, yleni henki heimon tään. Kuuhut
kulki taivahalla kuusten lomitse kurkistain — kansa se kasvoi
kuusten alla, käköset oksilla kukkui vain.

———

Koittipa vihdoin Wäinämön aika, Suomeni sankari-aika,

tietojen, taitojen, taikojen aika, laulujen, urhojen aika, jolloin
suur' oli Suomeni valta, laaja laulun ja soiton maa: Vienan
päältä ja Valdain alta Suvannon suurten vetten taa. Jolloin
Suomeni kansa sorja ollut ei lännen, ei idän orja, jolloin
Kalevan kalpa löi ja leimusi tietäjän taika.

Kauvas valliten merta kahta Permin purret kulki, kauvas

kaartaen Pohjanlahta Kainuu satamat sulki, loitos Karjalan
kannel kaikui, kauvas kantoi Jäämin jous, Taara-huutoja
rannat raikui, kussa Yösalon purret sous. Silloin suur' oli
Suomen kansa, Suomen leijona voimassansa, silloin heimoni
kunniaa koko Pohjola julisti julki.

Josp' ois astunut silloin miesi, kansani yhtehen tuonut,

temmannut miekkasi, rastinut tiesi, valtasi vaajat luonut! Josp'
ois Wäinämö harmaapäinen silloin tarttunut valtikkaan, taikka
lähtenyt Lemminkäinen sukunsa suurena kulkemaan, —
noussut heimonsa herttuaksi, veljeskansojen kaitsijaksi,
Wäinän rannoilta Ruijan suulle torvien soida suonut!
 Vaan ei kutsua kuullut kansa, raikunut torven tahti,
Wäinämö soitteli kanneltansa, impiä ajeli Ahti; eip' on astunut
silloin miestä heimoni onnea ohjaamaan, kansa ei tietänyt
Kalevan tiestä, veli ei tuntenut veikkoaan. Perman rannalle
Norja nousi, Väinän virtoja vieras sousi, Kainuun hautasi
kansojen yö — vaipui heimoni mahti.

—————

Tullut oli koolle kansa tuomaan touko-uhrejaan vetten,

vaarain partahilta kautta suuren Suomenmaan, tullut oli
nuoret, vanhat vakkojansa tuomahan, pyhän Päivänvuoren
päällä Ukon maljan juomahan. Tuolla tammipuiden alla urhot
vanhat tarinoi, täällä kummun tanterella nuori kansa karkeloi;
siellä istui Ilmariset, haastoi Wäinöt harmahat, täällä leikki
Lemminkäiset, astui Ainot armahat. Meri aaltos vaaran alla,
päivä paistoi päällä veen, käkö kukkui kultasuulla, tammet
huojui hiljakseen, rastas lauloi rannan puussa, sotka salmen
suussa sous silloin heimon valtavanhin tarttui maljahan ja
nous:

"Terve kevät Pohjolahan, Suomehen suvinen päivä,

terve lämmin lounatuuli, terve kumpuen käkönen!
Terve veet sinertämähän, viidakot vihertämähän,
terve maahan marjan varret, terve kukkaset keolle!

Paista päivä näille maille, näien peltojen perille,

nosta maa makoamasta, Luojan nurmi nukkumasta,
pane korret korttumahan, sekä varret varttumahan,
tuhansin neniä nosta, saoin haaroja hajota,
kynnöstämme, kylvöstämme! Eip' on maa mehua
puutu, voimoa Kalevan pelto, kun lie armo antajista,
 lupa luonnon tyttäristä. Oi Ukko ylijumala, hattarojen
hallitsija, iätä iästä pilvi, nosta lonka luotehesta,
vihmo vettä taivahasta, mettä pilvistä pirota orahille
nouseville, touoille tohiseville!"

Näinpä lausui heimon vanhin, kansa Ukon maljan joi,

vanhat haastoi harvaksensa, nuori kansa karkeloi; käkö
kukkui kultasuulla, tammet huojui hiljakseen, meri aaltos
vaaran alla, päivä paistoi päällä veen.

Silloin nousi outo purje ulko-ulapalta,

laiva kohti Suomen saarta laski lännen alta;
urhot tuota ensin luuli allin ampujaksi,
naiset kaupin haahdeksi ja saksan haljakaksi.

Katsottihin, vuotettihin, niin jo nousi toinen,

nousi toinen, nousi kolmas purje yhden moinen.
Vaan kun koko taivon ranta välkkyi valkealta,
silloin ties he vainolaisen tulleen lännen alta.

Silloin vanha vaiti jäi ja lakkas laulu nuoren, kotihinsa kansa

riensi päältä Päivänvuoren; kohta joka kukkulalta vainoliekit
nousi, kautta suurten Suomen vetten sotapurret sousi.

Tänne miehet, tänne miekat, tänne tapparat

tuliset! Lännen jouset, lännen joukot tuulen siivillä
tulevat, pistävät tulehen pirtit, kylät polttavat poroksi,
vievät viljat, kullat, helmet, kassapäistä
kaunehimmat.

Kaiva kullat kuusten alle, aarnihautoihin hopeat,

rahat kätke raunioihin, viljat purnuhun pudota, mutta
 kaunot kassapääsi saattaos salon sisähän, sinikorven
kainalohon, piilohon pimentopirtin! Elä itke, Suomen
impi, elä, äitini, valita!

Ei sinua vieras viene, koske koura vainolaisen,

kunis on kultasi elossa, poikasi kädessä pontta,
urhoja salolla Suomen, kalpoja Kalevan maassa!

Tänne miehet, tänne miekat, tänne tapparat

tuliset! Lännen joukot, lännen jouset tuulen siivillä
tulevat!

Anna tulla turmamiehen, saapua salakavalan! On

meillä oluet pantu, vara vierahan varattu: jouset
kaikki jäntehessä suun suloksi lännen sulhon, kaikki
tapparat tanassa pään menoksi merisen miehen!

Saapui kansa Auran suulle, poikki Pohjan tiestä, saapui sata

jousikättä, tuhat miekkamiestä, mutta taakse taistokentän
Päivän kukkulalle nousi Suomen suuret noidat suurten puiden
alle.

Päivä näin kun vuoteltihin, niin jo vieno ilta

saattoi luokse lännen laivat taivon rantehilta,
nousi Ruotsi rantamalle, nousi rautarinnoin,
läksi länsi taistelohon, läksi uskoninnoin.

Jop'on jouset jännittyi ja suihki sulkanuolet,

päivä peittyi vasamoihin, yöhön Pohjan puolet;
Lappi laaja katsomahan kaaloi tuota kummaa,
Turja kaikki kammostui jo talven yötä tummaa.
 Iski mies jo miestä vasten, miekka vasten miekkaa,
kalskui kalpa, raikui rauta, huuhtoi hurme hiekkaa;
silloin valkes Pohjan yö jo säilän säihkynnästä,
revontulten roihun näki Lappi etelästä.

Vaan ei miekoin, miehin yksin sotaa käyty siellä,

käytiin sotaa sanalla ja miettehellä, miellä,
taidon kalvat tahkottiin ja tiedon linnat luotiin,
kansanhengen kaikki voimat taistelohon tuotiin.

Tuolta takaa taistokentän tammikummultansa

lauloi Suomen suuret noidat tenholaulujansa,
lauloi pillat piilostaan ja turmat Tuonelastaan,
Turjan tuulet, Hornan henget vainolaista vastaan.

Mutta heitä vastapäätä loisti lännen valta,

välkkyi suuri kultaristi vaahderkunnahalta.
Siellä helkkyi helmivyöt ja Herran kuvat kulki —
siellä munkit Henrik piispan piirihinsä sulki.

Aina jatkui leikki julma, miesten leppä juoksi,

rynnättihin, väistyttihin, vaihtui luode, vuoksi.
Harveta jo Ruotsin joukot alkoi sieltä täältä —
silloin nousi outo lieska Päivänvaaran päältä.

Kuinka lie se syttynytkin? Liekö loihtijalta

kipunainen kirvonnunna tulustaulan alta,
vaiko — niinkuin kansa kertoo kirkon legendasta —
Ukon uhripuuhun iski liekki taivahasta.

Kuinka lie! Mut kauhu valtas Suomen miesten mielet,

kun he huomas vaaran päältä tuimat tulen kielet,
kauhu hiipi kalvan päihin, tunki miesten tarmoon, —
toiset pyrki pakohon ja toiset turvas armoon.

Näinpä loppui taisto suuri, taisto Wäinön taian,

näinpä päättyi päivä armas, päivä Wäinön aian.
Eikä vielä sammununna ollut soihtu ruskon,
kun jo Suomi Kupittaalla otti uuden uskon.

(Eino Leino: Tarina suuresta tammesta.)

HÄVISIVÄTKÖ HALTIJAT?

Tämmöinen on tarina.

Muinaisina aikoina tulivat valloittajat tähän maahan, tulivat kiiltävin

peitsin, kirjavin kypärin, sotakunniassa loistavaa ristiä edellänsä
kantaen. Kalevan miehet nousivat jumaliansa puolustamaan, kylät
paloivat, ryskyen kaatuivat pyhät lehdot. Valloittajan miekka ja tuli
voitti. Vastahakoinen kansa karkoitettiin korpiin kuolemaan, mutta
heikko kansa kastettiin uusien herrain alamaisiksi.

Kansoja valloitetaan ja kukistetaan, ihmisiä karkoitetaan ja

nöyryytetään, mutta kukistuvatko jumalat, ja voidaanko jumaliakin
karkoittaa? Vai minne joutuivat Ilmattaret, minne Päivättäret,
Kuuttaret, minne Ahti Vellamoineen, minne Tapio sinipiikoinensa?
Minne kaikki ne haltijat, jotka ihmiskieltä taitaen olivat ilman pitkiltä
pihoilta ihmiselle puhuneet, jotka meren hyrskyissä hänelle
miehuutta laulaneet, jotka tähtiöinä hänen haaveitansa herättäneet,
tai metsäsalojen poluilla hänen runomielensä virittäneet ja hänen
yksinäisyytensä ikävän vaihtaneet riemuun?

Jos miehet hävisivät, hävisivätkö jumalat?

 Eivät hävinneet, kaikki elävät vielä tänä päivänä, joka meille
paistaa.

Valloittajain tietämättä, tahtomatta, oli hurmeiseen maahan

istutetun ristin mukana seurannut myös Totuuden Henki, joka ei ole
koskaan tulen ja miekan kanssa liittoa tehnyt.

Maahan tultuansa se kutsui kansan hylkäämät jumalat eteensä ja

puhui heille näin:

"Minun nimessäni menköön kerran kaikki takaisin, mikä tulella ja

miekalla rakennettu on, niin että joka on ottanut, se on kerran
antava, ja joka on ryöstänyt, se on kerran jälleen lahjoittava, ja joka
on herraksi tullut, on itsensä palvelijaksi tekevä. Eikä se ole miekalla
ja tulella takaisin menevä, vaan minun enkelieni avulla. Katso, minä
teen teidät enkeleikseni, ja teidän puheenne olkoon jälleen ihmisille
kuuluva, kunnes kaikki täyttyy ja minä näen kahtia hajonneen
ihmiskuntani jälleen yhtyneenä."

Näin puhui totuuden henki.

Ja antoi enkeleille aikaa tuhannen vuotta, ja antoi heille äänen,

joka hiljaisena kuin aamutuuli soi ihmisten tunnoissa.

Se ääni soi vielä tänäkin päivänä, joka meille paistaa.

(Arvid Järnefelt: Maaemon lapsia.)

JUMALAN KAULAKORISTE JA PYHÄ
OLAVI.

Suomen suurin pyhimys oli Pyhä Henrikki. Lähinnä häntä oli Olavi.
Hän oli aikanaan pohjolan yhteinen pyhimys, jonka jäännösten luo
Norjan Nidarosiin Suomestakin tehtiin toivioretkiä ja jolle täällä
monet kirkot oli pyhitetty.

Tarussa "Jumalan kaulakoristeesta" on hänen nimensä jo mainittu

Perman maahan tehdyn retken yhteydessä. Mutta jo sitä ennenkin
oli hän tullut tekemisiin suomalaisten kanssa. Keväällä v. 1008 hän
purjehti omasta maastaan Norjasta itäänpäin ja saapui viikinkinä
Viron Saarenmaahan. Matka tapahtui Suomen saariston kautta ja
siitä yli Viron rannoille. Siellä tulivat virolaiset häntä vastaan. Hän
voitti heidät ja hävitti maan.

Virosta palasi Olavi takaisin Suomeen ja raastoi saalista sen

rannikoilta. Kun hän nousi maihin, pakeni kaikki kansa metsiin,
vieden omaisuutensa mukanaan. Kuningas ajoi heitä takaa ja saapui
metsien läpi erääseen viljeltyyn laaksoon. Siellä anasti hän tavaraa,
mutta ei ottanut ihmisiä sotavangeiksi. Iltasella palasi kuningas
laivoilleen. Kun hän miehineen taas oli joutunut metsään, riensi
kansaa kaikilta tahoilta häntä vastaan, ampuen ja ahdistaen heitä.
Kuningas kehoitti miehiään pitämään puoliaan ja taistelemaan niin
hyvin kuin voivat, mutta se ei ollut niinkään helppoa, sillä
suomalaiset hakivat metsien suojaa. Ennenkun kuningas oli päässyt
siitä ulos, oli hän menettänyt paljon miehiä ja monet olivat tulleet
haavoitetuiksi. Vasta illan tultua oli hän päässyt laivojensa luo. Yöllä
loihtivat suomalaiset myrskyn ja merenkäynnin, mutta kuningas
nostatti ankkurit ja luovaili mantereen läheisyydessä. Kuninkaan onni
oli suurempi kuin suomalaisten loitsutaito: hänen onnistui yöllä
luovia ulos merelle. Mutta suomalaisten sotajoukko seurasi maata
myöten rantaa pitkin niin kauan kuin kuninkaan laivat olivat
näkyvissä.

Tämän tulevan pyhimyksen retki Suomeen ei siis ollut mikään

ristiretki, ainoastaan tavallinen viikinkien ryöstöretki, eikä hänellä,
niinkuin myöhemmin Eerikki kuninkaalla, ollut saarnaajia ja kastajia
mukanaan. Mutta jo noin sata vuotta tämän retkensä jälkeen häntä
palveltiin pyhimyksenä Suomessa. Suomen kenties vanhin kirkko,
Lemböten kirkko Lemlannissa Ahvenanmaalla, joka lienee rakennettu
v. 1100, siis ennen Eerikki kuninkaan ristiretkeä, oli pyhitetty Pyhälle
Olaville.

Olavi oli vasta kerrotun retkensä aikana vielä pakana. Muutamia

vuosia myöhemmin hän omisti kristinuskon ja alkoi levittää sitä
maassaan. Tanskan kuningas Knut, sama, jonka luo Tore Hund
pakeni, sittenkun oli menettänyt kaulakoristeen ja muun
omaisuutensa Olavin miehille, karkoitti hänet kuitenkin maasta.
Koettaessaan valloittaa sitä takaisin, joutui hän Stiklestadin
tappelussa v. 1030 taisteluun kansalaisiaan, norjalaisia talonpoikia
vastaan, jotka olivat menneet maansa vihollisen, Knut kuninkaan
puolelle. Ennen tätä taistelua olivat Olavin vastustajat — niin kertoo
vanha taru — tulleet koolle päättämään, kuinka taistelu oli
järjestettävä ja kuka oli tuleva sen johtajaksi. Joku ehdottaa: "Sinä,
Tore Hund, sovit hyvin johtamaan taistelua Olavia vastaan. Sinulla on
tarpeeksi syytä siihen; onhan sinulla kostettavanasi sekä monen
sukulaisesi kuolema että se, kun hän pakotti sinut pakenemaan ja
jättämään hänelle kaiken omaisuutesi. Voiko sinulle tarjoutua
parempaa tilaisuutta kostaa kaikki kärsimäsi häpeä?" — Tähän
vastasi Tore Hund: "En rohkene minä nostaa merkkiä kuningas
Olavia vastaan enkä ruveta tämän sotajoukon johtajaksi. Kaikki
täällä eivät minua tottelisi. Mutta ei kenenkään ole tarvis muistuttaa
minua kaikesta, minkä olen kostoa velkaa Olaville. Olen valinnut
miehistäni yksitoista, kaikkein parhaat ja minä ajattelen, ett'emme
tule jättämään muille miekan mittelemistä Olavin kanssa, jos meille
siihen tilaisuutta tarjoutuu."

Rivit järjestyivät. Tore Hund miehineen asettui eturintamaan.

Oli mies, jonka nimi oli Torsten, laivanrakentaja, kauppias ja

taitava seppä, suuri ja väkevä sotilas. Hän oli joutunut vihoihin
kuninkaan kanssa, ja tämä oli ottanut häneltä hänen rakentamansa
suuren ja uuden laivan rikosten sovittajaisiksi. Hän meni nyt
rintamaan Tore Hundin luo ja sanoi: "Tässä tahdon olla seurassasi,
Tore, sillä jos tapaan Olavin, aion olla ensimäinen käymään hänen
kimppuunsa, jos vain pääsen häneen käsiksi." Tore otti vastaan
Torstenin, joka liittyi hänen seurueeseensa.

Kun molemmat sotajoukot olivat niin lähellä toisiaan, että miehet

tunsivat toisensa, astui Tore Hund miehineen esiin ja huusi:
"Eteenpäin, eteenpäin, talonpojat!" Nämä päästivät sotahuutonsa ja
ampuivat nuoliaan ja heittivät keihäitään. Myöskin kuninkaan miehet
päästivät sotahuutonsa ja kiihoittivat sitten toisiaan sanoilla, joita
heille oli opetettu: "Eteenpäin, eteenpäin, sotamiehet, ristin miehet,
kuninkaan miehet!"

Ilma oli ihana ja aurinko paistoi kirkkaasti. Mutta kun taistelu alkoi,
tuli punerrus taivaalle ja auringon eteen ja vähän aikaa oli niin pimeä
kuin yöllä. Olavi kuningas oli koonnut miehensä kukkulalle, ja he
hyökkäsivät talonpoikain kimppuun niin rajusti, että näiden rintama
taipui niin, että kuninkaan joukon rintama oli siinä, missä
talonpoikain viimeiset miehet olivat seisoneet. Taistelu oli ankara.
Kuningas kävi itse rajusti käsikahakkaan. Hän iski Torea
olkapääähän, mutta miekka ei purrut. Hän oli eräällä retkellään
lappalaisten luo teettänyt itselleen ja miehilleen poronnahkaturkit,
siten loihditut, että aseet purivat niihin vähemmän kuin rautapaitaan.
Oli vain niin kuin olisi pöly suitsunnut poronturkista. Tästä laulaa
runoniekka:

Hyvä hallitsija itse huomasi, kuinka taikataitoisen Lapin

tehoisa suojus varjeli väkevän Toren hengen.

Nyt kävi Tore kuninkaan kimppuun, ja he iskivät vuoroin muutamia

kertoja, mutta kuninkaan miekka ei pystynyt poronnahkaan. Toren
käsi kumminkin haavoittui.

Silloin sanoi kuningas eräälle sotilaalleen, jonka nimi oli Björn:

"Lyö sitä koiraa [Hund = koira], koska ei rauta häneen pure." Björn
pyöräytti kirvestä kädessään ja iski sen hamaralla. Isku sattui
olkapäähän ja oli niin voimakas, että Tore horjahti. Silloin iski Torsten
laivanrakentaja kirveellään Olavia ja isku sattui vasempaan jalkaan
yläpuolelle polven. Torsten sai samassa itse kuoliniskun, mutta
kuningas nojautui erääseen kiveen, heitti pois miekkansa ja rukoili
Jumalalta apua. Silloin pisti Tore Hund häntä keihäällään; pisto sattui
rautapaidan alle ja tunkeutui vatsaan. Vielä sai kuningas kaulaansa
iskun eräältä toiselta mieheltä. Nämä kolme haavaa tuottivat
kuoleman kuningas Olaville.

Niinpiankun hän oli kaatunut, kaatuivat melkein kaikki muutkin,

jotka olivat olleet hänen seurassaan.

Kun taistelu oli päättynyt, meni Tore Hund kuninkaan ruumiin luo,
hoiti sitä, laskien sen maahan, oikoen jäsenet ja levittäen vaatteen
sen yli. Kun hän pyyhki veren kuninkaan kasvoista, olivat ne niin
ihanat, että posket punoittivat, niinkuin hän olisi nukkunut, mutta
paljoa helakammin kuin ennen hänen eläessään. Kuninkaan verta
sattui Toren käteen ja sitä juoksi ranteen yli, jossa oli haava, eikä
siihen enää tarvittu mitään sidettä, niin pian se parani. Tore itse
todisti tämän, kun kuningas Olavin pyhyys tuli kaiken kansan tietoon,
ja hän oli kaikista kuningasta vastaan olleista suurmiehistä
ensimäinen todistamaan hänen pyhyyttään. Illan tultua kantoi
muuan talonpoika poikineen vainajan ruumiin autioon majaan, pesi
sen, kääri liinoihin ja kätki oksien alle. Yöllä haki majasta suojaa
sokea mies. Siinä haparoidessaan tunsi hän jotain kosteata
sormissaan ja kun hän sattumalta kosketti niillä silmäluomiaan, sai
hän yhtäkkiä näkönsä. Olavin viaton veri oli saanut aikaan tämän
ihmeen.

Talonpoika kaivoi sitten salaa ruumiin maahan Nid-virran rannalle.

Olavin kuoltua talonpojat valitsivat kuninkaakseen tanskalaisen

kuninkaan pojan. Ei kestänyt kuitenkaan kauan, ennenkun mielet
muuttuivat. Talonpojat alkoivat katua sitä, että vieraan valloittajan
puolella olivat taistelleet niin hurskasta ja kelvollista omamaista
kuningasta vastaan kuin Olavi kuningas oli ollut.
 Hänen vihamiehensä ja surmaajansa katuivat sitä, että olivat
aiheuttaneet hänen kuolemansa. Vähän jälkeen Olavin surman lähti
Tore maasta ja matkusti Jerusalemiin, josta ei koskaan palannut. Kun
vuosi oli kulunut kuninkaan kuolemasta, kaivettiin hänen ruumiinsa
maasta. Silloin nähtiin taas ihmeitä tapahtuneen. Olavi oli punakka ja
ihana ja hänen tukkansa ja kyntensä olivat kasvaneet. Hiukset
leikattiin ja pantiin tulisille hiilille, mutta tuli ei kyennyt niitä
kärventämäänkään. Piispa julisti Olavin pyhäksi mieheksi ja hänen
arkkunsa asetettiin Nidarosin Klemens-kirkon pääalttarille, jonne
sitten kaikilta haaroilta alkoi tulvia toivioretkeläisiä. Sittemmin
rakennettiin uusi komea kirkko sille paikalle, jossa pyhimys oli jonkun
aikaa ollut haudattuna ja johon maasta oli pulpahtanut ihmeitä
tekevä lähde.

Maine Olavin pyhyydestä ja hänen haudallaan tapahtuvista

ihmeistä levisi pian kaikkiin pohjoismaihin, myöskin Suomeen.
Dominikaanein ensimäinen luostari Suomessa sai hänestä nimensä.
Olavin nimeen kastettiin täällä lukematon joukko lapsia. Monet
monituiset kirkot pyhitettiin Pyhälle Olaville. Pyhä Olavi ja pyhä
Eerikki kuvattiin usein rinnakkain keskiajan kirkkomaalauksissa. He
olivat rinnakkain Ruotsin valtakunnan lipussa ja molemmilla
marttyyrikuninkailla on yhteinen alttari Upsalan tuomiokirkossa.
Olavin kuva tavataan Ahvenanmaan, Uudenmaan ja Satakunnan
sineteissä. Kun marski Torkel Knuutinpoika oli voittanut pakanalliset
karjalaiset ja rakentanut valtansa turvaksi Viipurin linnan, nimitti hän
sen lujimman tornin, jota eivät tuli eivätkä hyökkäykset tähän
päivään asti ole saaneet kukistumaan, Pyhän Olavin torniksi. Kun
venäläiset vuonna 1495 hyökkäsivät linnan kimppuun, kohtasivat he
siellä Knut Possen miehineen, jotka levittivät heidän eteensä Pyhän
Eerikin ja Pyhän Olavin liput ja voittivat. Ja kun kallioiselle saarelle
Savon sydämeen ensin rakennettiin puinen varustus ja sitten kivinen
linna, uskottiin se pyhän Olavin suojeltavaksi ja sai nimen Olavin
linna. Sekin linna seisoo vielä ja on yksi pohjolan kauneimpia. Pyhä
Olavi osasi suojella omansa.

Mutta pyhä Olavi ei ollut ainoastaan sodassa suojelija. Hänen

turvissaan kokoontui myöskin rauhan väki juhliansa viettämään. Oli
paikkoja maassamme, m.m. Ulvilassa, joissa veljeskunnat eli kiltat
olivat omistaneet Olavin pyhimyksekseen. Hänen nimeensä
sitoutuivat veljeskuntien jäsenet, maljoja juoden, auttamaan toisiaan
ja hankkimaan toisilleen oikeutta. Vanha sananlasku sanoo: "Olli
outoja tekee", s.o. ihmeitä; jo se osoittaa, mitä pyhimyksen usko
Suomessakin oli saanut aikaan, arvattavasti taudeista parantaen ja
hädässä auttaen.

Pyhimyksen kunniaksi vietettiin uhrijuhlia, teurastettiin "Olavin

lampaita" niin kaukana kuin Savon sydämessä ja niin myöhään kuin
1800-luvulla. Karjalassa oli tapana, että ensimäinen keväällä
syntynyt vuona, "Olavin villavuona", pidettiin koko kesän
keritsemättä aina Olavin messuun saakka, jolloin se suurin
juhlallisuuksin teurastettiin ja sitten pantiin toimeen pidot. Jokaiseen
sellaiseen juhlaan osaa ottaneen tuli sitoutua kuolemaansa saakka
noudattamaan tätä tapaa ja siis ylläpitämään sitä jälkeläisiäänkin
varten. Lammasta teurastettaessa ei saanut sen luita rikkoa. —
Kalvolan kirkossa säilytetty Olavin kuva kuletettiin kerran salaa pois,
mutta siitä oli tarun mukaan seurauksena se, että pellot lakkasivat
kasvamasta. Kun kuva saatiin takaisin, kasvoi viljakin vanhan
teränsä. Tämä tapahtui luterilaisella ajalla. Mutta vielä niinkin
myöhään kuin 1872 olivat kalvolaiset kiintyneet tähän kirkkonsa
vanhaan suojelijaan siihen määrin, että kun he lahjoittivat kirkkonsa
muut vanhat pyhimyskuvat Helsingin historialliselle museolle —
Olavin kuvaa ei annettu.
 Näin laajalle oli siis Olavin palvelus levinnyt Suomen kansaan ja
näin syvälle siinä juurtunut. Tuo vanha viikinki, joka ensin oli tullut
tänne ryöstäen ja hävittäen, saapui tänne sitten hyväntekijänä ja
armon antajana sodan ja rauhan asioissa. Tavallaanhan se oli
Perman vanha Jumala, joka toimitti hänet tänne. Tore Hundhan
surmasi Olavi kuninkaan kostoksi siitä, että tämä oli ottanut häneltä
sen kaulakoristeen, minkä hän oli anastanut Karlelta, joka taas oli
ryöstänyt sen Perman suomalaisten jumalan kaulasta hänen
pyhäkössään. Mutta ryöstöretki Perman maahan oli tapahtunut Olavi
kuninkaan alkuunpanosta ja hänelle oli lopulta joutunut Jumalan
kaulakoriste. Se oli siis suomalaisten pyhäkössä tapahtunut
häväistys, joka aiheutti Olavin kuoleman. Mutta hänen kuolemansa
teki hänestä ensin marttyyrin ja sitten pyhimyksen Suomessakin.
Suomalaisten yhden heimon pyhä Vienan rannalla oli siis lopulta
antanut pyhän heidän toiselle heimolleen Auran rannoilla ja Saimaan
saarilla.

(Juhani Aho.)
TARINA TURUN SYNNYSTÄ.

Jeesus tuntemattomana vaelsi maan päällä ja kylästä kylään kulki

saarnaten hyvyyden ja rakkauden oppia ja tapasi ihmiset kuuroiksi
sille, mitä hän heille opetti, niin saapui hän murheellisena kunnaalle,
jonka juurella hän näki miehen seisovan lähteen luona.
Huomaamatta Jeesusta vihelsi mies ja lehdikosta tuli nuori orivarsa
hänen luokseen. Tälle mies alkoi puhella:

— Heposeni, nytpä meille on tullut eteemme pitkä tie ja

vaivaloinen työ, mutta jos sinä sen kestät, kestän minäkin.

Ja mies hyväili hevostaan ja syötteli sille kädestään heiniä, jotka

rehevinä kasvoivat lähteen partaalla.

— Mies, anna minulle suojaa pääni päälle, sanoi Jeesus lähestyen

miestä, sillä olen uupunut, jalkani ovat tien kivistä tulleet verisiksi, ja
metsän pensaat ovat repineet pukuni.

— Antaisin sinulle suojan ja jakaisin oloni kanssasi, koska näytät

olevan yhtä yksinäinen kuin minäkin, vastasi mies. Mutta katso
tuonne, tupani on poltettu, niittyni on tallattu, viljani sotkettu.
Minulla ei ole mitään muuta kuin tämä hepo ja tämä lähde tässä,
joita voin omikseni sanoa. Juo lähteestä, ja se on sinua virvoittava.
Tämä lähde on pohjattoman syvä, ja sen vesi on aina kylmää.

— Mistä tiedät sen pohjattomaksi? kysyi Jeesus.

— Olenhan koettanut sitä mitata, mutta kivi, jonka köyteen sidoin

ja lähteeseen laskin, ei tavannutkaan pohjaa. Kerran tahdoin minä
samoin mitata taivaan kannen korkeutta, jännitin vahvimman
jouseni, otin nuoleni sulituimman. Korkealle kohosi nuoli, mutta
putosi taas, Yhtä korkea kuin taivas, yhtä syvä on tämä lähde. Ja sen
siunaus on suuri. Katso nurmea, kuinka se tässä iloitsee ja kukkasia
kauniisti kantaa. Niin tahdon, että muuallakin olisi. Ja siksi olen
päättänyt johtaa tästä uoman etemmäksi.

— Sinä tahdot tuottaa iloa muillekin. Ovatko he siis sinulle iloa

tuottaneet?

— Eivät.

— Eivätkö ole tahtoneet?

— Eivät ole ymmärtäneet sitä tehdä. Mutta miksi en minä sitä

tekisi heille?

— Ja miten aiot tuon uoman laatia?

— Siitä juuri puhelin heponi kanssa ja kysyin jaksaako se. Minä

aion valjastaa heponi auran eteen ja lähteen reunasta kyntää syvän
vaon kauas, niin kauas, että se vihdoin mereen ulottuu.

— Miksi tuon kaiken tekisit?

 — Jotta maa vihannoisi muuallakin niinkuin täällä, jotta vilja
kasvaisi runsaampana, kun se imisi maasta kosteutta.

Ja hän talutti hevosen auran eteen ja valjasti sen.

Ja Jeesus lähestyi hepoa, ja tämä silloin hirnui hiljaa ja kuin

hyväillen hankasi päätään hänen olkapäähänsä.

Kun mies sen näki, sanoi hän:

— Sinä olet hyvä ihminen, koska luontokappaleet sinua rakastavat!

Lähde minun kanssani matkalleni!

Ja Jeesus astui auran vieressä ja hänen kätensä lepäsi hevon

kaulalla. Syvälle maahan painui aura miehen vankkojen hartioiden
painosta, ja hopeisena juovana seurasi lähteen vesi heidän
luomaansa uomaa pitkin. Ja hepo veti, sen lihakset pingoittuivat ja
lujasti painuivat kaviot maahan. Kun se uupui, niin pani Jeesus
kätensä sen kuonon eteen ja hyväili hiljaa sen turpaa, ja hepo tunsi
syövänsä paratiisin yrttitarhan voimakkaasti tuoksuvaa ruohoa.

Ja vako tuli yhä pitemmäksi, ja jo kaukaa kimalteli veden välkkyvä

nauha.

— Tästä on suorin tie merta kohden, sanoi Jeesus viitaten

eteensä.

— Minä tiedän sen kyllä, vastasi mies, mutta tehkäämme kierros

tuonne, jossa näet nuo laakeat pellot ja niityt. Maa on siellä hyvää,
mutta kuivuu helposti helteessä ja silloin laiho surkastuu. Sinne
johdattakaamme kulkumme.

— Jos väsyt, sillä kierros on pitkä.

 — Minun täytyy jaksaa.

— Asuuko tuon pellon äyräällä ystäväsi?

— Ei minulla ole ystäviä. Hän se poltti minun taloni ja anasti

tavarani, mutta ajattelisinko sitä nyt, kun hänen pellolleen voin
johtaa lähteen veden.

Pitkän kierroksen tehtyä oli vako kynnetty lakeiden peltojen halki.

Kotvasen kuluttua virkkoi taas mies:

— Tuon ison kunnaan takana on kylä, ja siellä äveriästä väkeä,

mutta heillä ei ole jokea, ei järveä. Kyntäkäämme vako sinnekin.

— Onko siellä sinulla ystäviä tai omaisia?

— Siellä asuvat lapseni, kaksi poikaani ja tyttäreni.

— Sinä rakastat heitä?

— Ovathan he minun lapsiani.

— Rakastavatko he sinua?

— Elä kysy vieras sitä, vaan kyntäkäämme vako sinne ja

odottakaamme yötä, jotta he eivät minua näkisi.

Kauan he kulkivat, mies painaen auraa syvälle maahan, jotta tulisi

vako virran juostavaksi, ja Jeesus taluttaen hepoa.

Vihdoin näkyi meren poukama, ja sitä kohden he riensivät. Kun he

sen saavuttivat, vaipui mies uupuneena maahan.
 — Kauaksi kotikummultani olen tullut, sanoi hän, kovin kauaksi,
mutta uoma on kynnetty, ja katso miten vesi tänne meitä seuraa. Se
virtaa yhä runsaampana ja levittää uomaansa. Sanoinhan minä, että
minun lähteeni oli pohjaton ja että sen vesi on loppumaton.

Ja virta kasvoi yhä leveämmäksi ja hedelmöittävänä se kulki

seutujen halki. Asukkaat sen varrella rakensivat veneitä ja kulkivat
virran suuhun, jonne purret mereltä saapuivat. Siinä he tasaisella
kentällä vaihtoivat tavaroita ja tekivät kauppaa turulla. Suojaksensa
he joen suuhun rakensivat linnankin ja aivan joen äyräälle korkean
kirkon.

Mies tätä katseli, ja hänen mielensä täytti hiljainen ylpeys. Mutta

jokea myöten tulivat ne, jotka olivat häntä vainonneet. Ja kun he
miehen näkivät, niin ottivat he hänet kiinni ja telkesivät tyrmään.
Siellä he hänet tahtoivat nälkään tappaa.

Mutta eräänä yönä, tuntiessaan jo kuoleman tulon, näki mies

äkkiä edessään matkatoverinsa.

— Minä näen, että sinä olet kilvoituksesi kestänyt ja että sinä

kaipaat lepoa, sanoi Jeesus.

— Katso, eivät he minulle ole tahtoneet pahaa tehdä, mutta he

eivät ole tietäneet, mitä he tekivät, sanoi mies. Auta minua, sillä olen
uupunut.

— Minä tahdon taluttaa sinut asuntooni. Siellä on monta

asuinsijaa. Punaisesta hongasta ovat sen seinät, ja valkoiset on siellä
lavitsat, ja iloisesti sen ikkunoista paistaa päivä.
 — Minä en voi näin likaisena sinun asuntoosi tulla, sillä pitkät ajat
olen tässä tyrmässä saanut virua, vastasi mies.

— Tule, ja minä tahdon sinut puhtaaksi pestä!

Ja he astuivat tyrmästä. Ja kun he ennättivät kauniille nurmelle,

kosketti Jeesus kädellään maata, ja katso, kirkas lähde kumpusi
heidän jalkojensa juuresta.

Kun Jeesus sen lähteen vedellä oli miehen puhtaaksi pessyt, sanoi
mies:

— Miksis minut asuntoosi veit? Enhän minä ole kelvollinen sinne

tulemaan. Minähän olen yksinkertainen talonpoika ja sinä olet
kuninkaan poika.

Mutta Jeesus hymyili ja sanoi:

— Autuaat ovat yksinkertaiset, sillä heidän on minun valtakuntani!

Ja näin syntyi Aurajoki, Turun kaupunki ja se lähde, jota he

Kupittaaksi nimittävät.

(Jalmari Finne: Ylioppilaita.)

PAAVIN ENSIMÄINEN SUOMEA
KOSKEVA KIRJE.

"Aleksanteri, sen niminen kolmas paavi, Upsalan arkkipiispalle ja

hänen alipiispoilleen sekä Guthorm jaarlille."

"Sangen kova ja haikea valitus on apostooliselle istuimelle tuotu,

että suomalaiset ('Phinni') aina, milloin vihollisten sotajoukko heitä
uhkaa, lupaavat pitää kristillisen uskon ja halukkaasti pyytävät
kristillisen lain saarnaajia ja opettajia, mutta sotajoukon mentyä
hylkäävät uskon, halveksivat ja kovin vainoovat saarnamiehiä. Sen
vuoksi, koska he siten näkyvät halventavan Jumalaa ja pilkkaavan
kristinuskoa, ja koska he sillä tavoin osoittautuvat kahdenkertaisiksi
kadotuksen lapsiksi, pitäen autuutenaan ja elämänään ainoastaan
maallisia asioita, taivaallisista huolimatta, eikä ole soveliasta, että
kristitty nimi on hädässä suojeluksena, vaikka sitä onnen aikana
halveksitaan ja hyljitään, niin kehoitamme ja käskemme teitä kaikkia,
että, voidaksemme vasta heidän petostansa ja viekkauttansa
viisaudella ja maltilla kavahtaa, ette anna heidän enää, jos pakko
tulee, turvautua teidän apuunne ja suojaanne, ell'eivät heitä teidän
haltuunne linnoituksiansa, jos heillä semmoisia on, taikka muutoin
antavat niin täydellisen takauksen ja vakuuden, ett'eivät vasta
millään tavoin saata jalkaansa peräyttää tai valppauttanne kiertää,
vaan pakoitetaan vahvasti pitämään ja säilyttämään kristillisen uskon
merkkejä, ett'eivät enää näyttäisi olevan niiden joukkoa, joista on
sanottu: hän sinua tunnustaa, niin kauan kuin teet hänelle hyvää.
Annettu Tusculum'ista Syysk. 9 p:nä (1171 tai 1172)."
VIRON VALLOITUS
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