materials

Article
Titanium Dioxide Thin Films Obtained by Atomic
Layer Deposition Promotes Osteoblasts’ Viability and
Differentiation Potential While Inhibiting Osteoclast
Activity—Potential Application for Osteoporotic
Bone Regeneration
Agnieszka Smieszek 1 , Aleksandra Seweryn 2, * , Klaudia Marcinkowska 1 ,
Mateusz Sikora 1 , Krystyna Lawniczak-Jablonska 2 , Bartlomiej. S. Witkowski 2 ,
Piotr Kuzmiuk 2 , Marek Godlewski 2 and Krzysztof Marycz 3,4, *
1 Department of Experimental Biology, Wroclaw University of Environmental and Life Sciences, Norwida St.
27 B, PL-50375 Wroclaw, Poland; agnieszka.smieszek@upwr.edu.pl (A.S.);
klaudia.marcinkowska@upwr.edu.pl (K.M.); mateusz.sikora@upwr.edu.pl (M.S.)
2 Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, PL-02668 Warsaw, Poland;
jablo@ifpan.edu.pl (K.L.-J.); bwitkow@ifpan.edu.pl (B.S.W.); kuzmiuk@ifpan.edu.pl (P.K.);
godlew@ifpan.edu.pl (M.G.)
3 International Institute of Translational Medicine, Jesionowa 11 Street, 55-124 Malin, Poland
4 Collegium Medicum, Institute of Medical Science, Cardinal Stefan Wyszynski University (UKSW),
Wóycickiego 1/3, 01-938 Warsaw, Poland
* Correspondence: aseweryn@ifpan.edu.pl (A.S.); krzysztof.marycz@upwr.edu.pl (K.M.)




Received: 18 September 2020; Accepted: 26 October 2020; Published: 28 October 2020


Abstract: Atomic layer deposition (ALD) technology has started to attract attention as an efficient
method for obtaining bioactive, ultrathin oxide coatings. In this study, using ALD, we have
created titanium dioxide (TiO2 ) layers. The coatings were characterised in terms of physicochemical
and biological properties. The chemical composition of coatings, as well as thickness, roughness,
wettability, was determined using XPS, XRD, XRR. Cytocompatibillity of ALD TiO2 coatings was
accessed applying model of mouse pre-osteoblast cell line MC3T3-E1. The accumulation of transcripts
essential for bone metabolism (both mRNA and miRNA) was determined using RT-qPCR. Obtained
ALD TiO2 coatings were characterised as amorphous and homogeneous. Cytocompatibility of
the layers was expressed by proper morphology and growth pattern of the osteoblasts, as well as
their increased viability, proliferative and metabolic activity. Simultaneously, we observed decreased
activity of osteoclasts. Obtained coatings promoted expression of Opn, Coll-1, miR-17 and miR-21
in MC3T3-E1 cells. The results are promising in terms of the potential application of TiO2 coatings
obtained by ALD in the field of orthopaedics, especially in terms of metabolic- and age-related bone
diseases, including osteoporosis.

Keywords: atomic layer deposition; titanium dioxide; ultrathin layers; oxide layers; TiO2 coating;
improved viability; osteogenic properties

1. Introduction
The coating of surgical implants is designed to improve their biocompatibility and bioactivity.
The promotion of bone healing and the restoration of tissue homeostasis are essential factors to be
considered when designing new coatings for bone regeneration. Much attention is paid to novel
modified coatings with improved biological activity that affects the metabolism of progenitor cells

Materials 2020, 13, 4817; doi:10.3390/ma13214817 www.mdpi.com/journal/materials

Materials 2020, 13, 4817 2 of 20

by enhancing their viability and proliferation, as well as supporting cellular adhesion and increasing
cellular differentiation. This aspect is crucial, especially in relation to bone metabolic disorders, such
as osteoporosis. A thorough analysis of cells’ response to a biomaterial surface can provide some
insight into the cellular mechanisms controlling bone metabolism and homeostasis. Insufficient
integration of bone tissue with the implant surface can cause the implant to be rejected, leading to
severe complications, most often requiring revision surgeries [1]. Thus, tailoring biocompatibility of
biomaterials is usually associated with modifying its surface in order to improve the cells’ adhesion,
proliferation, and tissue-specific differentiation. Mechanical modifications are aimed at introducing
changes in the material’s topography to achieve optimal cell adhesion to the surface, while chemical
methods, for example those associated with anodising the titanium surface, yield nanotubes that
improve the biological and anti-microbial properties of the biomaterial [2]. Furthermore, sol-gel
methods were shown to improve the corrosion resistance of metal substrates and to enhance
the osteogenic differentiation of progenitor cells [3,4]. In addition, magnetron sputtering is a useful tool
for biofunctionalization of implant surfaces. However, it recently has been shown that the TiO2 obtained
by ALD technology provides better anti-corrosion properties independent of surface topography in
comparison to sputtered TiO2 [5]. It is also possible to obtain the high quality layer of TiO2 with
a Pulsed Laser Deposition (PLD) technique. PLD allows for depositing the films with variable porosity
and density [6].
Recently, there has been a growing interest in the application of atomic layer deposition (ALD)
technology for tissue-engineering applications and improving implants’ surfaces. ALD technology
allows us to fabricate ultrathin, highly uniform, and reproducible coverings with a broad range of
potential biological applications thanks to their biomimetic features [7]. Additionally, conformal
growth provides the possibility for functionalising the multidimensional surfaces [8]. Furthermore,
ALD technology can be used for temperature sensible materials that support tissue regeneration, e.g.,
polymer-based scaffolds, or composites. It was reported that homogenous ALD layers can be created
even at room temperature, which significantly extends their application in terms of scaffold modification
and the functionalisation of biomolecules, as well as other temperature-sensitive nanoparticles [9]. For
instance, ALD technology was previously used for the deposition of titanium dioxide (TiO2 ) coverings
onto tobacco mosaic virus (TMV) and ferritin. The application of ALD yielded pores and channels with
diameters less than 4 nm on the TMV, while nanotubes were fabricated with ferritin molecules [10].
The applicability of ALD technology is emerging in the field of bioengineering and regenerative
medicine, especially in the light of the significant advantages of this method over other techniques used
for depositing oxide coatings onto sensitive substrates. Previously, ALD technology was used to obtain
uniform TiO2 coatings with controllable thickness, not exceeding 2 nm. The coatings were deposited
on porous materials and three-dimensional objects, which indicated their high potential for application
in the functionalisation of implants designed for dentistry and orthopaedics [11]. However, Liu et
al. for the first time used ALD technology to obtain TiO2 nano-coatings with anti-bacterial properties
and high bioactivity. Their study showed a wide range of anti-microbial efficacy of ALD TiO2 coatings
that inhibit the growth of gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli), and
antibiotic-resistant bacteria (MRSA). Moreover, it was shown that ALD TiO2 coatings have a potential
selective function, promoting osteoblasts while suppressing fibroblast adhesion and proliferation. This
feature is of the utmost importance for orthopaedic implants that are designed to minimise fibrous
tissue formation and simultaneously maximise the formation of functional bone tissue [12].
In addition, a study by Yang et al. indicated further potential biomedical application of TiO2
coatings, showing that these nano-layers can be deposited on Mg-Zn alloy stents to enhance human
coronary artery endothelial cell adhesion and growth. The study revealed that an optimised processing
temperature control of the ALD TiO2 coatings is essential in order to achieve the proper biological
function of the biomaterial. Yang et al. have indicated that coatings deposited at 150 ◦ C have a greater
potential to promote the proliferation of endothelial cells than coatings deposited at 200 ◦ C [13].
Furthermore, Basiaga et al. indicated that the mechanical properties of TiO2 coatings obtained by
Materials 2020, 13, 4817 3 of 20

ALD technology strictly depend on the number of cycles during the process of deposition [14]. This
information is also of practical importance in terms of the application of ALD coatings for designing
implants, both for bone regeneration as well as for contact with blood, such as coronary stents.
Recently, Motola et al. showed the possibility to enhance the functionality of the Ti surface. They
investigated flat and nanotubular interfaces modified with ALD processes and considered the influence
of osteoblast, fibroblast, and neuroblast cells’ growth and proliferation [15]. Thin TiO2 coatings
obtained by photocatalytic patterning can also be improved by flower-like hierarchical Au structures
that promote the adhesion and proper growth of the osteoblast cells [16]. Moreover, the TiO2 coatings
covered with Au nanoparticles may significantly improve their photocatalytic activity [17] and increase
their potential application for example as a sensitive detectors of 17β-estradiol [6].
The response of cells to the contact surface is induced with both chemical and physical properties.
Wettability, roughness, and isotropic qualities of the materials are an important factor which can
determine cell response to the solid state surface and should be taken into account when designing
multifunctional coatings [18]. Mendonca et al. analyze the influence of nanoscale roughness and
chemical composition on the osteogenesis gene expression [19]. Given the emerging importance of
ALD technology in preparing TiO2 coatings for contact with bone tissue, we aimed to obtain TiO2
coatings that regulate the activity of both bone-forming and bone-resorbing cells. In the current
research, we were able to produce homogenous coatings at low temperatures with a thickness of 90 nm.
We have investigated their cytocompatibility using a model of mice pre-osteoblasts (MC3T3-E1 cell
line) as well as a co-culture with pre-osteoclasts (i.e., 4B12 cell line). We have established the influence
of TiO2 coatings obtained by ALD and MC3T3-E10 s viability, proliferative potential, metabolic activity,
morphology, and growth pattern. In the co-culture model of osteoblasts and osteoclasts, we have also
evaluated the influence of TiO2 coatings on the expression of markers associated with bone metabolism.
The biomarkers were evaluated at the messenger ribonucleic acid (mRNA) and micro ribonucleic acid
miRNA levels. This study shows, for the first time, the modulatory effect of TiO2 coatings obtained by
ALD on the osteoblast–osteoclast coupling.

2. Materials and Methods

2.1. Substrate
The thin TiO2 films were deposited on glass coverslips 13 mm in diameter for biological testing
and physicochemical characterisation. The adhesion and quality of the layer deposited by the ALD
may be limited by possible surface contamination. This is why all the substrates were thoroughly
washed in an ultrasonic cleaner and then dried before the ALD process. The first bath took place in
isopropanol, the next two in deionised water. All wash cycles were carried out for 5 min in a temperature
bath of 37 ◦ C. Subsequently, nitrogen gas with a purity of 5.0 was used to dry the substrates after
the cleaning process.

2.2. ALD Growth Method

The deposition of TiO2 film was carried out in a Savannah-100 Cambridge NanoTech (now Veeco)
reactor. Two precursors were alternately introduced into the reaction chamber. First, an organic
precursor was selected as a metal precursor: Tetrakis(dimethyloamino)titanium (CAS no.: 3275-24-9,
Strem Chemicals, Inc., Newburyport, MA, USA). The second one, an oxygen precursor, was deionised
water. The purging phase after each dose of precursors was carried out with nitrogen gas with a purity
of 6.0. The precursor feeding was as follows: 0.2 s dose of metal precursors, 3 s waiting phase,15 s
pulse of purging gas, 0.04 s pulse of oxygen precursor, 3 s waiting phase, and another purging gas dose
of 15 s. This protocol was repeated 1220 times. The process was carried out under stable temperature
(100 ◦ C) and pressure (66 Pa). Additionally, the titanium precursor was preheated to 70 ◦ C. The heaters
of the reactor chamber and precursor were turned on an hour before the start of the process to ensure
that the correct temperature was reached and stabilised and held during the layer growth. After ALD
Materials 2020, 13, 4817 4 of 20

processes, the samples were vacuum packed and transferred to further biological and physical research.
There were no additional cleaning procedures before the physical measurements.

2.3. Analysis of the Physicochemical Properties of the Coatings

The X-ray diffraction (XRD) assays were used to determine the crystallinity of the TiO2 films.
The panalytical X’Pert Pro MRD diffractometer was used in the XRR analysis. The generated X-ray
radiation was at a wavelength of 1.54056 Å. The Pixcel detector and Parallel Plate Collimator with
0.4-rad Soller slits and a 0.18-deg divergence slit were applied. Based on Parratt’s theory, Panalytical
software [20] was used to determine the thickness, electron density, and roughness of the resulting
coating. The rate of decay of the X-ray signal can determine the roughness of the surface: the amplitude
of the oscillations observed is related to the thickness of the layer on the surface of the substrate and
the width of the oscillations changes with the electrons’ density of the material.
The Scanning Electron Microscopy (SEM) investigations were performed using a Hitachi SU-70
system. Images were taken at 15 kV of accelerating voltage using detector of secondary electrons.
To determine the chemical compounds of the layers obtained, X-ray photoelectron spectroscopy
(XPS) measurements were taken. The XPS measurements were carried out according to a previously
described protocol [21] using a Scienta R4000 hemispherical analyser (pass energy: 200 eV) and Al Kα
(1486.7 eV) with non-monochromatic excitation. The analysis of full width at half maximum (FWHM)
of the 4f7/2 Au line was measured under the same experimental conditions as TiO2 layer was 1.1 eV.
The energy scale was calibrated by setting the C 1s line at the position of 285.6 eV [21].
The wettability of the surface is a crucial parameter in determining the biocompatibility of
the material. We assessed the hydrophilic/hydrophobic properties of biologically tested substrates
by measuring the contact angle of a drop of water deposited on the surface. A goniometer OCA 25
from DataPhysik was utilised to obtain the contact angle for fluid on the TiO2 surface deposited onto
a coverslip. The test fluid was water. The measurement was performed under normal conditions
(temperature: 25 ◦ C; air humidity: 50%). As a reference, the wettability measurement on an uncoated
coverslip was taken. All tests were repeated three times at different sites on the samples, a TiO2 -coated
coverslip, and an uncoated coverslip. The result for links and rights contact angle was taken into account.

2.4. Evaluation of the Cytocompatibility of TiO2 Coatings Obtained by ALD

Culture of pre-osteoblastic mice cell line: MC3T3-E1 cell line cells were maintained in Minimum
Essential Media Alpha (MEM-α, Gibco™ Thermo Fisher Scientific, Warsaw, Poland). The culture
conditions have been thoroughly described by other authors [21,22]. The medium was supplemented
with 10% Fetal Bovine Serum, (FBS, Sigma Aldrich, Munich, Germany) and changed every 2–3 days.
The cells were cultured at constant, aseptic conditions in a CO2 incubator at 37 ◦ C and 95% humidity.
The cultures were passaged using a trypsin solution (StableCell Trypsin, Sigma Aldrich, Munich,
Germany) after reaching 90% confluence. The protocol was previously described in detail [22]. Before
the trypsinisation step, the cells were washed in Hanks’ Balanced Salt Solution (HBSS) without calcium
or magnesium. Th cells were passaged using trypsin solutions and common protocols [22]. For
the digestion, 3 mL of trypsin solution was added to a T75 culture flask (Nunc, USA), and the cells
were incubated for 5 min at 37 ◦ C in a CO2 incubator. The cells used for these experiments were at
passage number 24 (p24). For the experiment, MC3T3-E1 cells were inoculated in 24-well plates at
a density equal to 2 × 105 cells per well. The cells were maintained in 0.5 mL of CGM (MEM-α with
10% FBS). The medium was refreshed 3 times per week.
The influence of TiO2 coatings obtained by ALD on the apoptosis profile: The apoptosis profile
was analysed using a MUSE Annexin V & Dead Cell Kit (Merck, Sigma-Aldrich, Poznan, Poland)
according to the manufacturer’s protocols and previously published information [22]. To put it briefly,
cells were detached with trypsin, centrifuged (5 min, 300× g), and diluted with 100 µL of PBS containing
1% FBS. The cell suspension and 100 µL of Annexin V/dead reagent were mixed in a 1.5-mL centrifuge
tube and incubated for 20 min at room temperature in the dark. Then, the cells were analysed using
Materials 2020, 13, 4817 5 of 20

a Muse Cell Analyser (Merck, Sigma-Aldrich, Poznan, Poland). The apoptotic cell distribution was
evaluated by identifying four populations: non-apoptotic (viable) cells: Annexin V (−), 7-AAD (−);
early apoptotic cells: Annexin V (+), 7-AAD (−); late apoptotic/dead cells: Annexin V (+), 7-AAD (+);
and dead cells: Annexin V (−) and 7-AAD (+).
The influence of TiO2 coatings obtained by ALD on cell proliferation: The proliferative activity
was evaluated using a MUSE Cell Cycle Kit (Merck, Sigma-Aldrich, Poznan, Poland) according
to the manufacturer’s instructions. A detailed protocol was published by other authors [22]. For
the analysis, the cells were collected after detachment from the culture flask with trypsin solution,
centrifuged (5 min, 300× g), washed with 1X PBS, fixed with 1 mL of 70% cold ethanol, and incubated
overnight. Then, the cells were centrifuged and washed, as described previously [22]. The cell pellet
was suspended in 200 µL of Muse Cell Cycle Reagent (Merck, Warsaw, Poland) and incubated at
room temperature for 30 min in the dark. The distribution of cells in G0/G1, S, and G2/M phases was
estimated using a Muse Cell Analyser (Merck, Sigma-Aldrich, Poznan, Poland).
The influence of TiO2 coatings obtained by ALD on the cells’ metabolic activity: The metabolic
activity of the cells was estimated using a TOX-8 resazurin-based in vitro Toxicology Assay Kit (Sigma
Aldrich, Munich, Germany) according to the manufacturer’s instructions and previously published
protocols [23]. The complete growth medium was replaced with fresh CGM supplemented with 10%
resazurin solution (Sigma Aldrich, Munich, Germany). The cells were incubated for 2 h at 37 ◦ C
in a CO2 incubator. Then, the supernatants were transferred to a 96-well plate (100 µL per well).
The absorbance was measured as indicated before [4,21,22]. The effect of TiO2 coatings on the cells’
metabolic activity was assessed after 24, 48, 72, 96, and 168 h of culture (not all data are shown).
The influence of TiO2 coatings obtained by ALD on mitochondrial potential: changes in
the mitochondrial potential of MC3T3-E1 cells cultured in TiO2 were monitored using a MUSE
MitoPotential Kit (Merck, Warsaw, Poland) according to the supplier’s protocols. The cells were
harvested by trypsinisation, centrifuged (5 min, 300× g), and suspended in 100 µL of Assay Buffer
(Merck, Warsaw, Poland). Next, 95 µL of MitoPotential working solution was added to the cell
suspension. Assay tubes were vortexed for 3 s and incubated for 20 min in a CO2 incubator at 37 ◦ C.
Then, 5 µL of Muse MitoPotential 7-AAD reagent (Merck, Warsaw, Poland) was added to the cell
suspension and the samples were incubated for 5 min at room temperature. The mitochondrial
potential was assessed using a Muse Cell Analyser (Merck, Warsaw, Poland) by the identification
of four populations: live cells with depolarised mitochondrial membrane: MitoPotential (-), 7-AAD
(-); live cells with intact mitochondrial membrane: MitoPotential (+), 7-AAD (-); dead cells with
depolarised mitochondrial membrane: MitoPotential (+), 7-AAD (+); and dead cells with disturbed
mitochondrial membrane potential: MitoPotential (-) and 7-AAD (+).
Determination of the TiO2 coatings’ obtained by ALD influence on cell morphology, ultrastructure,
and adhesion rate: The morphology and ultrastructure of the MC3T3-E1 cells were analysed after
72 h of culturing. The detailed protocol of culture staining and preparation for confocal imaging was
described by other authors [24]. The specimens were analysed using a confocal microscope (Leica TCS
SPE, Leica Microsystems, KAWA.SKA Sp. z o.o., Zalesie Gorne, Poland) and the microphotographs
obtained were then analysed using Fiji (ImageJ 1.52n, Wayne Rasband, National Institute of Health,
Bethesda, Maryland, USA), as described previously [22]. The figures presented herein were obtained
using the maximum intensity projection (Z-projection). In addition, the ultrastructure of cultures was
examined with a scanning electron microscope (SEM, Zeiss Evo LS 15, Oberkochen, Germany). Before
SEM analysis, the cells were fixed (in 4% paraformaldehyde [PFA] as described above) and dehydrated
in an ethanol series (concentrations from 50% to 100%, each incubation for 5 min). The specimens were
sputtered with gold and observed using an SE1 detector at 10 kV of filament tension [4]. The adhesion
rate of the MC3T3-E1 cells was determined using the protocol published by Huang et al. [25] and used
previously [26,27].
Co–culture with pre-osteoclastic cell line 4B12: The osteoclast precursor cell line 4B12 was kindly
provided by Shigeru Amano from the Department of Oral Biology and Tissue Engineering, Meikai
Materials 2020, 13, 4817 6 of 20

University School of Dentistry. The detailed description of the culture method was published before [22].
The cells used in the experiment were at passage number 28 (p28). The 4B12 cells were cultured with
pre-seeded MC3T3 cells. For co-culturing, the 4B12 cells were inoculated at a density equal to 3.5 × 104
in a chamber of an 8-µm Transwell system membrane (Corning, Biokom, Warsaw, Poland). The cells
were maintained in 0.3 mL of α-MEM with 10% FBS and 30% CSCM; half of the medium was changed
3 times per week. Th edetailed protocol of co-culturing was described elsewhere [22]. The invasion of
4B12 was determined based on SEM images.
The influence of TiO2 coatings obtained by ALD on osteogenesis marker gene expression
and miRNA levels: To determine the mRNA and miRNA levels, the experimental cultures were
homogenised using 1 mL of Extrazol® (Blirt DNA, Gdansk, Poland). The protocol for total RNA isolation
was performed according to the manufacturer’s instructions and the modified phenol-chloroform
method described by Chomczyński and Sacchi [28]. The resulting RNA was diluted in DEPC-treated
water. The quantity and purity of RNA specimens were determined spectrophotometrically at 260-
and 280-nm wavelengths (Epoch, Biotek, Bad Friedrichshall, Germany). Before reverse transcription,
the total RNA obtained (500 ng) was purified using DNAse I (PrecisionDNAse, PrimerDesign, BLIRT
S.A. Gdansk, Poland). The reverse transcription was performed using a Tetro cDNA Synthesis Kit
(Bioline Reagents Limited, London, UK). Both processes were performed according to well-established
protocols [14,15]. The DNA digestion and cDNA synthesis were carried out in a T100 Thermal
Cycler (Bio-Rad, Hercules, CA, USA). The resulting matrices were used for RT-qPCR analysis using
a SensiFAST SYBR® &Fluorescein Kit (Bioline Reagents Ltd., London, UK). The final reaction volume
was 10 ul, where 1 ul of cDNA was used and the concentration of primers was 0.5 µM. Quantitative
PCR was performed in a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA,
USA). The details of the protocols and the reaction conditions were described previously [22].
Additionally, to evaluate the miRNA levels, cDNA was also synthesised using 375 ng of total RNA
with a Mir-X™ miRNA First-Strand Synthesis Kit (Takara Bio Europe, Saint-Germainen, Laye, France)
as described by other authors [21]. The primer sequences are summarised in Table S1. All qPCR
reactions were carried out in at least three repetitions. The relative values of gene expression were
determined with the RQMAX algorithm as described previously [29]; for normalisation, Gapdh
(glyceraldehyde 3-phosphatedehydrogenase) was used as a reference gene and U6snRNA (Takara Bio
Europe, Saint-Germainen, Laye, France) was used to determine the levels of miRNA.

2.5. Statistical Analysis

The results obtained during in vitro studies are presented as the mean of a minimum of three trials.
The means are presented with standard deviations (± SD). Statistical comparisons between the data
were assessed by one-way analysis of variance (ANOVA) and unpaired Student’s t-test. The results
were analysed using GraphPadPrism 5 software (La Jolla, CA, USA). Differences with a probability of
p < 0.05 were considered to be statistically significant.

3. Results

3.1. Physicochemical Properties of TiO2 Coatings Obtained by ALD

The thin layer of titanium dioxide was successfully deposited onto glass substrates for biological
investigations and physicochemical tests.
The XPS measurement of O 1s, C 1s, and Ti 2p lines correspond to a content of 22.1 at.% of titanium
(Ti), 52.3 at.% of oxygen (O), and 25.6 at.% of carbon (C) on the sample surface. No other elements
were found (Figure 1a). The relatively high adventitious carbon originated mostly from contamination
of the surface due to air exposition of the sample before XPS measurements and was used for energy
scale calibration (C 1s binding energy [BE] 285.6 eV). The stoichiometry ratio in the oxide compound is
determined by the content of the metal ions and the oxygen ions. The ratio of O:Ti content was 2.4,
confirming the growth of an amorphous layer with stoichiometry close to TiO2 . The excess oxygen may
Materials 2020, 13, 4817 7 of 20

have resulted from the adsorption of water and carbon oxide on the surface. The result was confirmed
by analysis of the oxygen 1s line Figure 1b). Two components fit this line well: the main component at
BE = 530.8 eV covers 86% of the line and the second component BE = 532 eV covers only 14%. Taking
into account only the main component at 530.8 eV of energy—which is close to the oxygen-binding
energy in TiO2 [30,31]—the corrected ratio of oxygen to titanium is 2.04. It confirms the formation of
an amorphous titanium dioxide with stoichiometry close to the ideal. The energy positions (459 eV
and 464 eV) and the separation in the Ti 2p spin-orbit doublet (5.6 eV) agree within the limit of energy
calibration error (± 0.2 eV) with the values reported in the reference table and other publications
(Figure 1c) [32]. Moreover, in the case when the many O defects are present and stoichiometry of ALD
layer is strongly changed, the presence of Ti+3 is observed in Ti 2p line as e.g., in [5].

Figure 1. The XPS data plot of TiO2 obtained by ALD on a glass substrate: (a) wide XPS spectra—only
lines of O, Ti, and C were detected, C 1s binding energy (BE) 285.6 eV was used for energy scale
calibration; (b) O1s experimental and fitting line; (c) Ti 2p experimental and fitting line.

The XRR measurement was performed on the coverslip deposited with the TiO2 coating as well
as on the coverslip. We observed a change in the surface roughness of the samples. The simulation of
experimental data indicates that the roughness of the surface of the coverslip is 0.7 nm (Figure 2b);
for the TiO2 coating, the roughness parameter was simulated to the value of 2.4 nm (Figure 2c).
The simulated density is equal to 3.88 g/cm3 , which is lower than the table data from bulk 4.23 g/cm3
titanium dioxide material. The thickness of the deposited coating was estimated to 90 nm.

Figure 2. The XRR data plots of TiO2 obtained by ALD on a glass substrate (green colour) and uncoated
coverslip (magenta colour): (a) the experimental line; (b) the fitting line (blue colour): for the coverslip
sample; (c) the fitting line for TiO2 on the coverslip sample (fit simulation made using Parratt’s theory).

To determine the quality of the coating, we performed SEM measurements. The images presented
in Figure 3 show a very high uniformity (Figure 3a) of the coating. No larger crystallites are visible on
the surface, which is characteristic of amorphous layers (Figure 3a,b).
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Figure 3. The SEM images of TiO2 obtained by ALD on Si substrate as a reference: cross-section view
(a)Figure
and 3.
Figure 3.The
top TheSEM
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(b). Images
images of
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at 15 kV
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ALD
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electrons.electrons.
secondary
Additionally, to confirm the amorphous phase of the coating, we performed X-ray diffraction
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our the layer report
previous also points
[33] to an
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other research structure.
[9]. The obtained result is in good agreement with our previous report [33] and
structure. The obtained result is in good agreement with our previous report [33] and other research [9].
other research
The evaluation [9].
evaluationof ofthe
thewettability
wettabilitydata data (Figure 4) shows that, in relation
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a
2
of the contact angle is for coverslip 62.9◦ and 93.3◦ for TiO2 sample. This finding indicates a more
value of the contact angle is for coverslip 62.9° and 93.3°
more substantial hydrophobic property of TiO2 compared to a pure coverslip. for TiO 2 sample. This finding indicates a
substantial hydrophobic
more substantial property
hydrophobic of TiO2ofcompared
property TiO2 compared to a pure
to a coverslip.
pure coverslip.

Figure Theimages
4. The
Figure 4. imagesofofwater
watercontact
contactangle
angle(wettability)
(wettability) measurement
measurement forfor
(a)(a) a coverslip
a coverslip andand (b) TiO
(b) TiO 2 2
on Figure
a 4. The
coverslip images
(CA leftof water contact
indicates angle
measured (wettability)
links’ contactmeasurement
angle, for
while CA(a) a coverslip
right and
indicates
on a coverslip (CA left indicates measured links’ contact angle, while CA right indicates measured (b) TiO
measured2

on a coverslip
right (CA left indicates measured links’ contact angle, while CA right indicates measured
right contact angle).
angle).
right contact angle).
3.2.
3.2. Cytocompatibility
Cytocompatibility ofofthe
theTiO
TiO2 2Coatings
CoatingsObtained
Obtained byby ALD
ALD
3.2. Cytocompatibility of the TiO2 Coatings Obtained by ALD
The
The analysis
analysis ofofcell
cellviability
viabilityindicated that
indicated thethe
that TiOTiO
2 coating exhibited non-toxic properties toward
2 coating exhibited non-toxic properties
the The analysis of (Figure
cell viability indicated thatsignificantly
the TiO2 coating exhibited non-toxic properties
toward the MC3T3-E1 cell line (Figure 5). The biomaterial significantly the
MC3T3-E1 cell line 5). The biomaterial improved viability
improved theof viability
preosteoblasts
of
toward
(Figure the Moreover,
4a,b). MC3T3-E1 we cell line (Figure 5). The apoptosis
biomaterial in significantly improved the viability of 2
preosteoblasts (Figure 4a,b).observed
Moreover, decreased
we observed decreasedMC3T3-E1
apoptosiscultures propagated
in MC3T3-E1 on TiO
cultures
preosteoblasts
obtained by on
ALD, (Figure 4a,b). Moreover, we observed decreased apoptosis in MC3T3-E1 cultures
propagated TiOthough
2 obtained thebydifferences
ALD, thoughwerethenot statistically
differences weresignificant (Figure
not statistically 5c).
significant (Figure
propagated on TiO2 obtained by ALD, though the differences were not statistically significant (Figure
5c).
5c).
Materials 2020, 13, 4817 9 of 20
Materials 2020, 13, x FOR PEER REVIEW 9 of 20

Figure 5. The cell viability and apoptosis profile in the control culture (TiOALD ALD coatings-) and
Figure 5. The cell viability and apoptosis profile in the control culture (TiO2 2 coatings-) and the
the culture propagated on TiO2 coatings (TiO2ALD coatings +; experimental culture): (a) representative
ALD
culture propagated on TiO2 coatings (TiO2 coatings +; experimental culture): (a) representative
graphs obtained during analysis, showing the distribution of cells on four populations: live
graphs obtained during analysis, showing the distribution of cells on four populations: live (Live—
(Live—bottom-left corner), early apoptotic (Early Apop.—bottom-right corner), late apoptotic (Late
bottom-left corner), early apoptotic (Early Apop.—bottom-right corner), late apoptotic (Late
Apop./Dead—upper-right corner), and dead (Dead—upper-left corner); results of statistical analysis
Apop./Dead—upper-right corner), and dead (Dead—upper-left corner); results of statistical analysis
showing a comparison of viable cells (b) and apoptotic cells (c) in control and experimental cultures
showing a comparison of viable cells (b) and apoptotic cells (c) in control and experimental cultures
(significant differences are marked with asterisks (* p < 0.05), non-significant results are marked as ns).
(significant differences are marked with asterisks (* p < 0.05), non-significant results are marked as
ns). analysis of the distribution of cells in the cell cycle revealed that preosteoblast MC3T3-E1 cells
The
cultured on TiO2 surfaces obtained by ALD (Figure 6) showed increased proliferative activity, which
The analysis of the distribution of cells in the cell cycle revealed that preosteoblast MC3T3-E1
was reflected in the accumulation of cells in the S phase (Figure 6a,c). Moreover, in cultures propagated
cells cultured on TiO2 surfaces obtained by ALD (Figure 6) showed increased proliferative activity,
on TiO2 coatings obtained by ALD, we observed more cells in the G2/M phase (Figure 6a,d). The shift
which was reflected in the accumulation of cells in the S phase (Figure 6a,c). Moreover, in cultures
of cells toward the G2/M phase was accompanied by fewer cells in the G0/G1 phase (Figure 6a,b).
propagated on TiO2 coatings obtained by ALD, we observed more cells in the G2/M phase (Figure
6a,d). The shift of cells toward the G2/M phase was accompanied by fewer cells in the G0/G1 phase
(Figure 6a,b).
Materials 2020, 13, x FOR PEER REVIEW 10 of 20

Materials 2020, 13, 4817 10 of 20

Materials 2020, 13, x FOR PEER REVIEW 10 of 20

Figure 6. The results of the analysis of the control culture (TiO2 ALD coatings-) and a culture propagated
on TiO2 coatings (TiO2 ALD coatings +; experimental culture), showing the distribution of cells in the
6. The results of thehistograms
analysis of show
the control culture (TiOof ALD coatings-) and a culture propagated
cellFigure
cycle: (a) Representative the distribution 2 MC3T3 cells in the cell cycle phase
Figure
on TiO 6. The
coatingsresults
(TiO of the
ALD analysis
coatingsof the
+; control
experimental culture (TiO
culture),2 ALD coatings-)
showing and
theG0/G1 a culture propagated
distribution of cells
2
under both culture conditions. 2 The cells were separated into three populations: phase (the left in
on
the TiO
cell 2 coatings
cycle: (a) (TiO 2 ALD coatings +; experimental culture), showing the distribution of cells in the
Representative histograms show the distribution of MC3T3 cells in the cell cycle phase
side), S phase (the middle), and G2/M phase (the right side). The results of statistical analysis revealed
cell cycle: (a) Representative histograms show the distribution of populations:
MC3T3 cells G0/G1
in the cell cycle phase
differences in cell distribution during the (b) G0/G1 phase, (c) S phase, and (d) G2/M phase. left
under both culture conditions. The cells were separated into three phase (the
under
side), S both culture
phase (the conditions.
middle), and The cells
G2/M phasewere(the separated
right into
side). Thethree populations:
results of G0/G1
statistical phase
analysis (the left
revealed
(significant differences are marked with asterisks (*** p < 0.001; ** p < 0.01; * p < 0.05).
side), S phase
differences (the
in cell middle), and
distribution G2/M
during thephase (the right
(b) G0/G1 side).
phase, (c) SThe results
phase, andof(d)
statistical analysis
G2/M phase. revealed
(significant
differences
Thedifferences
Alamar Blue
in cell
are marked distribution
with asterisks
test indicated (*** p <metabolic
during
increased
the (b) G0/G1< 0.01; * pof< MC3T3-E1
phase,
0.001; ** p activity (c) S phase,
0.05). and (d) G2/M phase.
cells cultured in the
(significant differences are marked with asterisks (*** p < 0.001; ** p < 0.01; * p < 0.05).
presence of TiO2 coatings (Figure 7). The MC3T3-E1 cells demonstrated a significantly faster
The Alamar Blue test indicated increased metabolic activity of MC3T3-E1 cells cultured in
metabolism in response to TiO2 coating obtained by ALD at the initial stage of culturing, i.e., after 24
The Alamar
the presence of TiOBlue test indicated
2 coatings (Figureincreased metabolic activity
7). The MC3T3-E1 of MC3T3-E1a cells
cells demonstrated culturedfaster
significantly in the
h of propagation (Figure 7a), but also after 72 h and 168 h of culturing (Figure 7c,e).
presence ofin TiO
metabolism 2 coatings
response to TiO(Figure
2 coating 7). The
obtained MC3T3-E1
by ALD at cells
the demonstrated
initial stage of a significantly
culturing, i.e., after faster
24 h
ofmetabolism
propagationin(Figure
response tobut
7a), TiOalso
2 coating
after obtained by ALD
72 h and 168 at the initial
h of culturing stage7c,e).
(Figure of culturing, i.e., after 24
h of propagation (Figure 7a), but also after 72 h and 168 h of culturing (Figure 7c,e).

Figure 7. The metabolic activity in the control culture (TiO2 ALD coatings-) and a culture propagated on
TiO2 . coatings (TiO2 ALD coatings +; experimental culture): comparative analysis of metabolic activity
after (a) 24 h, (b) 48 h, (c) 72 h, (d) 96 h, and (e) 168 h of propagation. (significant differences are marked
Figure 7. The metabolic activity in the control culture (TiO2 ALD coatings-) and a culture propagated on
p < 0.001; ** p < 0.01; * p < 0.05), non-significant results are marked as ns).
with asterisks (***ALD
TiO2. coatings (TiO2 coatings +; experimental culture): comparative analysis of metabolic activity
after (a)
The 24 h,
analysis(b) 48 h, (c) morphology
of cells 72 h, (d) 96 h,showed
and (e) 168 h of propagation.
no significant (significant
influence of aTiO differenceson arethe cells’
Figure 7. The metabolic activity in the control culture (TiO2 ALD coatings-) and 2 coating
culture propagated on
marked with asterisks (*** p < 0.001; ** p < 0.01; * p < 0.05), non-significant results are marked as ns).
morphology or mitochondrial
TiO2. coatings network
(TiO2 ALD coatings development
+; experimental (Figure
culture): 8a). Our observations
comparative revealedactivity
analysis of metabolic that both
the control
after (a)cultures
24 h, (b)and the(c)experimental
48 h, 72 h, (d) 96 h,cultures hadh aofproperly
and (e) 168 expanded
propagation. network
(significant of theare
differences actin
marked with asterisks (*** p < 0.001; ** p < 0.01; * p < 0.05), non-significant results are marked as ns).
Materials 2020, 13, x FOR PEER REVIEW 11 of 20

The analysis of cells morphology showed no significant influence of TiO2 coating on the cells’
Materials 2020, 13, 4817 11 of 20
morphology or mitochondrial network development (Figure 8a). Our observations revealed that both
the control cultures and the experimental cultures had a properly expanded network of the actin
cytoskeleton, maintained
cytoskeleton, maintainedcell–cell
cell–cellcontact,
contact, andand appropriately
appropriately adhered
adhered to substrate.
to the the substrate. Moreover,
Moreover, both
both
the the MC3T3-E1
MC3T3-E1 cells cultured
cells cultured on coverslip
on a plain a plain coverslip
and those and those
coated bycoated
ALD withby ALD
TiO2 with TiO2 had well-
had well-developed
developed mitochondrial
mitochondrial networks networks
(Figure 8a). (Figure
The8a). The MC3T3-E1
MC3T3-E1 cells maintained
cells maintained proper proper morphology
morphology and
and ultrastructure
ultrastructure in the
in both both the control
control and experimental
and experimental cultures.cultures. In addition,
In addition, no occurrence
no occurrence of apoptoticof
apoptotic
bodies wasbodies
observed wasinobserved
the MC3T3 in cultures
the MC3T3 cultures
(Figure (Figure 8b). we
8b). Nevertheless, Nevertheless, we found cells
found that MC3T3-E1 that
MC3T3-E1 cells
propagated ontopropagated
TiO2 surfaces onto
wereTiO 2 surfaces were
characterised by characterised by lower
lower mitochondrial mitochondrial
potential potential
in comparison to
in comparison
the to thethis
control cultures; control cultures;
difference wasthis difference
statistically was statistically
significant (Figure significant
8c,d). At the(Figure 8c,d).we
same time, At did
the
same
not time, we
observe did notdifferences
significant observe significant differences
in the percentage in the
of total percentage
depolarised cellsofbetween
total depolarised
the controlcells
and
between the control and experimental cultures propagated
experimental cultures propagated on TiO2 layers (Figure 8c,e). on TiO 2 layers (Figure 8c,e).

Figure
Figure 8. Theresults
8. The resultsofofthe
theultrastructural
ultrastructuralanalysis
analysis
of of MC3T3
MC3T3 cells
cells in in
thethe control
control culture
culture (TiO
(TiO 2 ALD
2
ALD coatings -) and a culture propagated on TiO coatings obtained by ALD (TiO ALD coatings
coatings -) and a culture propagated on TiO2 coatings 2 obtained by ALD (TiO2 2ALD coatings +;
+; experimental
experimental culture):
culture): (a) confocal
(a) confocal imaging
imaging showing
showing nucleinuclei organisation
organisation (blue—stained
(blue—stained with
with DAPI),
DAPI), an actin cytoskeleton (green—Atto 488 Phalloidin), and the distribution of the
an actin cytoskeleton (green—Atto 488 Phalloidin), and the distribution of the mitochondrial networkmitochondrial
network (red—mitoRed).
(red—mitoRed). Thewere
The pictures pictures were captured
captured under magnification
under magnification equal
equal to 630× to 630×
(scale bar (scale bar
= 40 μm);
= 40 µm); (b) the ultrastructure of the MC3T3-E1 cells. The pictures were captured
(b) the ultrastructure of the MC3T3-E1 cells. The pictures were captured using SEM under 500× using SEM
under 500× magnification
magnification (scale bar = (scale
100 μm = 100
barand µm and magnification
magnification equal (c)
equal to 2500×); to 2500×);
Dot-plots(c)presenting
Dot-plots
presenting
distributiondistribution
of cells basedof cells based on mitochondrial
on mitochondrial membranemembrane
potential. potential.
Cells wereCells were separated
separated into four
into four populations: live (Live—bottom-right corner), live with the depolarised mitochondrial
populations: live (Live—bottom-right corner), live with the depolarised mitochondrial membrane
membrane (Depolarized/Live—bottom-left corner), dead with the depolarised mitochondrial membrane
(Depolarized/Live—bottom-left corner), dead with the depolarised mitochondrial membrane
(Depolarised/Dead—upper-left corner), and dead (Dead—upper-right corner). The results of
(Depolarised/Dead—upper-left corner), and dead (Dead—upper-right corner). The results of the
the statistical analysis showing the percentage of cells with (d) high mitochondrial potential and
statistical analysis showing the percentage of cells with (d) high mitochondrial potential and (e) total
(e) total depolarised cells. (significant differences are marked with asterisks (* p < 0.05), non-significant
depolarised cells. (significant differences are marked with asterisks (* p < 0.05), non-significant results
results are marked as ns).
are marked as ns).
The analysis of preosteoclast invasion properties indicated that the number of preosteoclasts
The analysis of preosteoclast invasion properties indicated that the number of preosteoclasts is
is significantly lower in the cultures of the preosteoblast MC3T3 propagated on a TiO2 coating,
significantly lower in the cultures of the preosteoblast MC3T3 propagated on a TiO2 coating, (Figure
(Figure 9a,b). In addition, the measurement of MC3T3-E1 adhesion indicated that TiO2 coatings
9a,b). In addition, the measurement of MC3T3-E1 adhesion indicated that TiO2 coatings promote
promote osteoblast attachment to surfaces. The results are in agreement with the increased metabolic
osteoblast attachment to surfaces. The results are in agreement with the increased metabolic activity
activity of MC3T3-E1 preosteoblasts propagated on a TiO2 layer, measured when they first interact
of MC3T3-E1 preosteoblasts propagated on a TiO2 layer, measured when they first interact with the
with the biomaterial, which strictly depends on the adhesive properties of the surface.
biomaterial, which strictly depends on the adhesive properties of the surface.
Materials 2020, 13, 4817 12 of 20
Materials 2020, 13, x FOR PEER REVIEW 12 of 20

Figure
Figure 9.9. The
The invasion
invasionof
ofpreosteoclasts
preosteoclastsininthe
thecontrol
controlco-culture (TiO
co-culture 2 -)2and
(TiO thethe
-) and culture propagated
culture on
propagated
TiO ALD
2 coatings
on TiO obtained by ALD (TiO2 2ALDcoatings+;
2 coatings obtained by ALD (TiO coatings+;experimental
experimentalco-culture):
co-culture): (a)(a) the
the representative
representative
pictures of MC3T3-E1 co-cultured with pre-osteoclasts. 4B12 cells were visualised and coloured
pictures of MC3T3-E1 co-cultured with pre-osteoclasts. 4B12 cells were visualised and coloured red
red (GNU Image Manipulation Program 2.10.18). The pictures were captured using SEM under
(GNU Image Manipulation Program 2.10.18). The pictures were captured using SEM under 500×
500× magnification (scale bar = 20 µm); (b) the results of statistical analysis showing the number of
magnification (scale bar = 20 µ m); (b) the results of statistical analysis showing the number of
preosteoclasts. (significant differences are marked with asterisks (** p < 0.01)).
preosteoclasts. (significant differences are marked with asterisks (** p < 0.01)).

The pro-osteogenic properties of the TiO2 coatings were confirmed by the expression of genes
The pro-osteogenic properties of the TiO2 coatings were confirmed by the expression of genes
involved in the process of osteogenesis and proper bone mineralisation. The analysis was performed
involved in the process of osteogenesis and proper bone mineralisation. The analysis was performed
for the MC3T3-E1 cell line, as well as for co-cultures of MC3T3-E1 with pre-osteoclastic cell line
for the MC3T3-E1 cell line, as well as for co-cultures of MC3T3-E1 with pre-osteoclastic cell line 4B12.
4B12. In the MC3T3-E1 cultures propagated on TiO2 surfaces, we observed a higher expression of late
In the MC3T3-E1 cultures propagated on TiO2 surfaces, we observed a higher expression of late
osteogenesis markers, such as osteopontin (Opn) and osteocalcin (Ocl) (Figure 10b,d). Simultaneously,
osteogenesis markers, such as osteopontin (Opn) and osteocalcin (Ocl) (Figure 10b,d).
the same cultures were characterised by lower levels of mRNA for other osteogenic markers, i.e.,
Simultaneously, the same cultures were characterised by lower levels of mRNA for other osteogenic
collagen type 1 (Coll-1) and runt-related transcription factor 2 (Runx2) (Figure 10a,c). Interestingly,
markers, i.e., collagen type 1 (Coll-1) and runt-related transcription factor 2 (Runx2) (Figure 10a,c).
the profile of osteogenic markers for MC3T3-E1 cultured with 4B12 was maintained in the cultures
Interestingly, the profile of osteogenic markers for MC3T3-E1 cultured with 4B12 was maintained in
propagated on the TiO2 coating. As a result of the paracrine effects of preosteoclasts, MC3T3-E1
the cultures propagated on the TiO2 coating. As a result of the paracrine effects of preosteoclasts,
cultured in control conditions had lower levels of osteogenic genes. Obtained results correspond with
MC3T3-E1 cultured in control conditions had lower levels of osteogenic genes. Obtained results
the increased invasiveness of 4B12 noted in the control cultures. In turn, the MC3T3-E1 propagated on
correspond with the increased invasiveness of 4B12 noted in the control cultures. In turn, the MC3T3-
TiO2 and in direct contact with 4B12 were characterised by a higher accumulation of transcripts for
E1 propagated on TiO2 and in direct contact with 4B12 were characterised by a higher accumulation
Opn, Ocl, and Runx2 (Figure 9b,c and Figure 10a–d).
of transcripts for Opn, Ocl, and Runx2 (Figures 9b,c and 10a–d).
 Materials
Materials 2020,
2020, 13,13, x FOR PEER REVIEW
4817 1313ofof
2020

Figure
Figure10.10.The
ThemRNA
mRNAexpression
expression of of genes
genes associated with osteogenic
osteogenic potential.
potential. The
Theanalysis
analysisofofthe
thecontrol
controlculture
culture(TiO ALD coatings -) and the culture propagated on TiO coatings (TiO ALD
(TiO22ALD coatings +;
coatings -) and the culture propagated TiO22coatings (TiO22 ALD coatings
+; experimental
experimental culture) examined
examined (a) (a)Coll-1,
Coll-1,(b) (b)Opn,
Opn,(c) (c)Runx-2,
Runx-2,andand(d)
(d)Ocl.
Ocl.The
Thetranscripts’
transcripts’profiles
profiles
were measured using the RT-qPCR technique. The relative quantification
were measured using the RT-qPCR technique. The relative quantification (RQ) was performed (RQ) was performed using
using
the RQMAX
the RQMAX method
method andandthe results
the resultsarearepresented
presented inin
a log scale.
a log (significant
scale. differences
(significant areare
differences marked
marked
with asterisks
with asterisks(*** p <p 0.001;
(*** < 0.001; p<
**** 0.01;* p* p<<0.05),
p <0.01; 0.05),non-significant
non-significantresults
resultsare
aremarked
markedasasns).
ns).

TheThelevels
levelsofofmicroRNAs
microRNAsinvolvedinvolvedininbone
bonemetabolism
metabolismwerewerealsoalsoaltered
alteredininresponse
responsetotoTiOTiO
2 2
coatings (Figure 11). In the MC3T3-E1 cells cultured
coatings (Figure 11). In the MC3T3-E1 cells cultured onto TiO onto TiO obtained by ALD surfaces,
2 2 obtained by ALD surfaces, we we
observed
observeda asignificantly
significantlyhigher
higher expression
expression of ofmiR-17
miR-17(Figure
(Figure11c)
11c)
andand miR-21(Figure
miR-21(Figure 11d),
11d), while
while miR-
miR-124 (Figure 11b) levels were lowered. This profile corresponds with mRNA
124 (Figure 11b) levels were lowered. This profile corresponds with mRNA levels for osteogenic levels for osteogenic
markers, i.e.,Opn
markers,i.e., Opnand andOcl,Ocl,showing
showingthat
thatthetheTiO 2 layer
TiO may provide pro-osteogenic conditions,
2 layer may provide pro-osteogenic conditions,
inducing
inducing differentiation
differentiation of MC3T3-E1 cells into
of MC3T3-E1 osteoblasts.
cells However, However,
into osteoblasts. the MC3T3-E1 the cells propagated
MC3T3-E1 cells
onpropagated
ALD covered with TiO samples and influenced by the paracrine activity
on ALD covered with TiO2 samples and influenced by the paracrine activity of
2 of preosteoclasts
showed significantly
preosteoclasts showed lower levels of miR-7
significantly lowerandlevels
miR-21,
of which
miR-7 are
andconsidered
miR-21, whichosteogenic miRNAs.
are considered
Nevertheless, the lower Nevertheless,
osteogenic miRNAs. levels of miRNAs
the promoting
lower levels osteoclast activity,
of miRNAs i.e., miR-7
promoting and miR-124,
osteoclast were
activity, i.e.,
significantly lower, a finding which also correlates with the decreased invasion
miR-7 and miR-124, were significantly lower, a finding which also correlates with the decreased of 4B12 preosteoclasts
ininvasion
this condition.
of 4B12 preosteoclasts in this condition.
 Materials 2020, 13, x FOR PEER REVIEW 14 of 20
Materials 2020, 13, 4817 14 of 20

Figure 11. The expression of miRNA associated with osteogenic potential. The analysis of the control
cultures (TiO2 ALD coatings -) and the cultures propagated on TiO2 coatings obtained by ALD (TiO2
Figure 11. The expression of miRNA associated with osteogenic potential. The analysis of the control
ALD coatings +; experimental culture) examined (a) miR-7, (b) miR-124, (c) miR-17, and (d) miR-21.
cultures (TiO2 ALD coatings -) and the cultures propagated on TiO2 coatings obtained by ALD (TiO2 ALD
The transcripts’ profiles were measured using the RT-qPCR technique. The relative quantification (RQ)
coatings +; experimental culture) examined (a) miR-7, (b) miR-124, (c) miR-17, and (d) miR-21. The
was performed using the RQMAX method, and the results are presented in a log scale. (significant
transcripts’ profiles were measured using the RT-qPCR technique. The relative quantification (RQ)
differences are marked with asterisks (*** p < 0.001; ** p < 0.01), non-significant results are marked as
was performed using the RQMAX method, and the results are presented in a log scale. (significant
ns).
differences are marked with asterisks (*** p < 0.001; ** p < 0.01), non-significant results are marked as
4. Discussion
ns).

Currently, much interest can be observed in the development and application of ALD technology
4. Discussion
as a method for creating bioactive coatings for orthopaedic implants. Mounting evidence indicates
Currently,
that ALD technologymuch interest
provides canoption
a new be observed in the development
for functionalising and application
the biomaterials’ of ALD
surface, improving
technology as a method for creating bioactive coatings for orthopaedic implants.
the metabolism of bone progenitor cells, and promoting osseointegration. In this study, we used Mounting evidence
indicates
ALD that ALD
technology technology
to obtain thin TiO provides a new optionby
2 films distinguished forselective
functionalising
biological theproperties:
biomaterials’ surface,
activating
pro-osteogenic signals and inhibiting the invasion of osteoclast precursors. TiO2 coatings study,
improving the metabolism of bone progenitor cells, and promoting osseointegration. In this had
we used ALD
previously beentechnology
described and to obtain thin TiO2 in
characterised films
termsdistinguished by selectiveproperties
of their antibacterial biological and
properties:
their
activating pro-osteogenic
cytocompatibility signals and
toward progenitor inhibiting
cells, including thepreosteoblasts
invasion of osteoclast precursors.
[1,2]. It was indicatedTiOthat
2 coatings
thin,
had previously been described and characterised in terms of their antibacterial
ALD-fabricated TiO2 meets the criteria of pro-osteogenic coatings that promote bone-forming cell properties and their
cytocompatibility
growth toward
and proliferation [12].progenitor
The quality cells,
of theincluding
TiO2 layers preosteoblasts[1,2].
obtained by ALDItmeets was indicated that thin,
the requirements
ofALD-fabricated TiO2 meets
coatings for implant the criteria
materials. A thin ofTiO pro-osteogenic
2 layer was coatings
deposited that
on promote
scaffolds bone-forming
made of titanium cell
growth and proliferation [12]. The quality of the TiO layers obtained by ALD
powder, mimicking the biological functions of the substrate. The TiO2 was coated on the porous
2 meets the requirements
of coatings
metallic for implant
biomaterial materials.
uniformly A thin
and with highTiO 2 layer
quality wasAdditionally,
[11]. deposited on TiO scaffolds made of titanium
2 films deposited on 316
powder, mimicking the biological functions of the substrate. The TiO was
LVM steel surfaces have been investigated mechanically. Basiaga et al. demonstrated that modifying
2 coated on the porous
metallic biomaterial uniformly and with high quality [11]. Additionally, TiO2 films deposited on 316
LVM steel surfaces have been investigated mechanically. Basiaga et al. demonstrated that modifying
Materials 2020, 13, 4817 15 of 20

the surface of vascular stents is possible; the mechanical properties of such layers—the thicknesses of
the layers—depend on the number of ALD cycles used in the process [14].
The ALD technique attracts attention as a promising technology that allows for tailored, unique
biocompatible coatings to be fabricated. However, the TiO2 coatings obtained by ALD and designed
for potential biomedical application are usually created in temperatures significantly higher than
100 ◦ C. For example, Liu et al. deposited TiO2 at 200 ◦ C [34], while Liu et al. created coatings
with ALD in the 120–190 ◦ C temperature range [35]. In this study, we performed the TiO2 growth
process at a temperature of 100 ◦ C. The selection of precursors, the metal precursor in particular, and
the temperature during the ALD growth process are the key parameters which determine the phase
composition of the thin TiO2 coating. The tetrakisdimethyloamino used by us in this study has a low
growth rate of TiO2 , indicating inefficient surface reactions. For comparison, the growth rate when
using tetrakisdimethyloamino of metals such as hafnium or zirconium is twice as high [33]. However,
despite the low growth rate, the high quality of the coating indicates the stoichiometry of this dioxide
was preserved. TiO2 occurs in various crystallographic phases—amorphous, rutile, anatase, and
brookite—or it can coexist in several phases [35]. The crystal phase of TiO2 obtained by ALD depends
strongly on the deposition temperature [9], while the amorphous layer is formed at low deposition
temperatures, anatase at medium temperatures, and rutile at the highest temperatures. It has also
been proven that crystallography influences the biological properties of the coating. For example,
Rossi et al. have shown that TiO2 coating which contains an anatase and rutile phase absorbing
the proteins from physiological fluids better [36]. The initial protein adhesion to the surface determines
the developmental phases of the cells (differentiation and proliferation) and whether osseointegration
is successful. Despite a lack of noticeable crystallographic order of the Ti and O atoms, we found
excellent osteogenic properties of the TiO2 coating.
An amorphous structure leads to better adhesion to the substrate as compared to the corresponding
crystalline layer [33]. Moreover, a low temperature of deposition is a significant advantage, allowing
such a coating to be applied on polymer surfaces that can change their structure at high temperatures.
Amorphous oxides obtained by low temperature are often far from stoichiometric. Park et al. showed
in their work the formation of crystallites with higher oxygen content while depositing TiO2 with
plasma-enhanced ALD. While the TiO1.6 layer was amorphous, the increased oxygen content (TiO1.7 )
resulted in the formation of crystallites [37]. In our study, we obtained amorphous TiO2 coating close
to ideal stoichiometry. The reason for this phenomena can be the fact that the generation of oxygen
vacancies was thermodynamically blocked at low temperatures during the growth process.
The TiO2 coating increased the hydrophobicity of the surface. This result is contrary to data
reported by Liu et al. They found a lower water contact angle with the deposition of an ALD layer.
However, in such a case, the wettability was determined by the substantial increase of surface roughness
rather than the surface chemistry. Cell adhesion is generally strongly correlated with the hydrophilic
properties of the materials. We found better cell adhesion despite the higher water contact angle. It
appears that their finding depended on the surface chemistry, and that the nearly ideal stoichiometry
may influence the biological properties of TiO2 .
In this study, we found that 90-nm TiO2 coatings obtained by ALD may promote proper bone
formation and may enhance the viability, proliferation, and metabolic activity of preosteoblasts. We
were able to determine the influence of TiO2 coatings obtained by ALD on both osteoblast and osteoclast
activity. We have indicated that TiO2 layers improve the metabolic activity and viability at the early
stages of cell–biomaterial contact and that they lesson the invasion of osteoclast progenitors. These
features are extremely desirable and are required for bone implant coverings, as they can ensure
the proper integration of biomaterials with bone tissue and can guarantee active bone remodelling. We
have also found that TiO2 coatings promote the adhesion of preosteoblasts and have the features of
a biomimetic structure, allowing for the control of cell–surface interaction.
The improved adhesion of osteoblast to TiO2 coverings was noted previously. For example,
Shokuhfar et al. reported that Ti surfaces treated with amorphous and crystalline TiO2 nanotube
Materials 2020, 13, 4817 16 of 20

are effective in increasing the number of attached MC3T3-E1 preosteoblasts. In addition, using SEM
and FIB analysis, Shokuhfaret al. provided direct evidence on the interlocked mechanism between
the cell and TiO2 . It was shown that osteoblasts growing on nanostructured TiO2 coatings create
filopodia extensions, increasing the contact area and resulting in better anchorage to the surfaces [38].
The increased adhesion of osteoblasts to TiO2 coatings was also described by Rivera-Chacon et al., who
explained this phenomenon by the selective absorbance of vitronectin and fibronectin by substrates
with nanostructures [39]. This finding partially explains our results, which indicate the improved
attachment of osteoblasts into the TiO2 layer and the inhibited invasion of osteoclasts. Vitronectin was
shown to promote osteoblast differentiation and activity, whilst concomitantly restraining osteoclast
differentiation and resorptive function [40].
The increased adhesion, as well as the improved proliferation and viability of progenitor cells,
ensures the guided regeneration of bone and the formation of functional tissue. In this study, we
showed that TiO2 coatings exert an anti-apoptotic effect towards preosteoblasts, significantly increasing
their viability and promoting cellular metabolism. Such features were described previously in relation
to the cytocompatibility of TiO2 coatings. TiO2 coatings obtained through ALD had been reported as
bioactive layers that modulate the metabolism of progenitor cells, affecting their osteogenic potential.
In this study, we confirmed that TiO2 coatings obtained by ALD activate transcripts associated with
preosteoblast differentiation into bone-forming cells. We found that TiO2 layers increased the mRNA
levels of osteopontin and osteocalcin in the pre-osteoblastic MC3T3-E1 cell line, which is in line with
the results presented by Vercellino et al., who showed that titanium dioxide nanostructured coatings
promote the differentiation of bone marrow stromal cells, elevating the expression of osteopontin and
osteocalcin [41].
In addition, we found that the gene expression pattern correlates with higher levels of regulatory
microRNAs, such as miR-17 and miR-21. It was previously reported that TiO2 -nanotube arrays regulate
the miRNA levels in human adipose-tissue-derived stem cells (hASCs) propagated under osteogenic
conditions. The increasing interest in microRNA involvement in the regulation of pro-osteogenic signals
also reinforces the studies on the effect of biomaterial and nanotopography-guided differentiation of
progenitor cells. Understanding mRNA–miRNA networks as an axis regulating the fate of progenitor
cells can be paramount when designing biomaterial-based therapies for metabolic disorders, including
osteoporosis. For example, it was previously reported by various groups, including ours, that miR-21
promotes osteogenesis, but also acts as a regulator of osteoclastogenesis and a promoter of osteoclast
differentiation [22,42]. Similarly, it was indicated that miR-17-5p improves cell proliferation and
osteoblastic differentiation of human multipotent stromal cells. Furthermore, it was shown that
decreased expression of miR-17-5p is correlated with worse clinical characteristics and poor survival
rate in patients with non-traumatic osteonecrosis [43]. Instead, miR-7 levels have not been thoroughly
described in terms of osteoblast biology, and it was found that it can be differentially expressed,
depending on bone metabolism [44,45]. It seems that the inhibition of miR-7 targeting the epidermal
growth factor receptor (EGFR) may inhibit the development of osteoporosis [45]. This conclusion is in
agreement with the profile of miR-124, which is an essential molecule regulating osteoclastogenesis.
The overexpression of miR-124 could inhibit osteoclastogenic differentiation of bone-marrow-derived
monocyte cells, indicating that the inhibition of miR-124 expression might be a potential therapeutic
strategy for the treatment of osteoporosis [46].
Pro-osteogenic properties of TiO2 layers obtained by ALD can also be expressed by decreased
mitochondrial membrane potential. This is the characteristic feature of differentiated MC3TC3-E1
osteoblasts, as described by Guntur et al. Moreover, a lack of a significant increase in mitochondrial
volume fraction during the differentiation of MC3T3-E1 cells to osteoblasts was also observed, which
can be explained by the fact that differentiated osteoblasts are not programmed to use oxidative
phosphorylation to supply their ATP demand [47]. Our results indicated that TiO2 coatings obtained
by ALD can play the role of a regulator mitochondrial adaptation and can exert anti-apoptotic
Materials 2020, 13, 4817 17 of 20

effects toward osteoblast precursors. This suggests that their potential application in metabolic- and
age-related bone diseases.

5. Conclusions
Nanoscale and biomimetic TiO2 coatings obtained by ALD have displayed promising
pro-osteogenic properties, activating the osteogenic biomarkers associated with proper bone
remodelling and regulating mitochondrial activity. We demonstrated that TiO2 coverings significantly
promote the adhesion of preosteoblast cells and inhibit the invasion of preosteoclasts, lowering
the levels of microRNAs (miR-7 and miR-124), which are crucial for osteoclast survival and maturation.
The TiO2 coatings obtained by ALD can be a suitable layer for enhancing the osteogenic properties and
biofunctionality of substrates used in the field of orthopaedics, especially in terms of metabolic- and
age-related bone diseases.

Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/13/21/4817/s1,

Figure S1: The XRD data of: TiO2 coating obtained by ALD on the cover slip (a), blank cover slip as a reference (b).
Table S1: The list of oligonucleotides used for determination of specific transcripts.
Author Contributions: Conceptualisation, A.S. (Agnieszka Smieszek), A.S. (Aleksandra Seweryn), M.G.;
methodology, A.S. (Agnieszka Smieszek), K.M. (Klaudia Marcinkowska), M.S., A.S. (Aleksandra Seweryn), B.S.W.,
P.K., K.L.-J.; software, A.S. (Agnieszka Smieszek), K.M. (Klaudia Marcinkowska), A.S. (Aleksandra Seweryn);
validation, A.S. (Agnieszka Smieszek); K.M. (Klaudia Marcinkowska)., A.S. (Aleksandra Seweryn), K.L.-J.; formal
analysis, A.S. (Agnieszka Smieszek), A.S. (Aleksandra Seweryn), K.L.-J., M.G.; investigation, K.M. (Klaudia
Marcinkowska), M.S, B.S.W., P.K.; resources, M.G., K.M. (Krzysztof Marycz); data curation, A.S. (Agnieszka
Smieszek), K.M. (Klaudia Marcinkowska), A.S. (Aleksandra Seweryn). K.L.-J., B.S.W.; writing—original draft
preparation, A.S. (Agnieszka Smieszek), A.S. (Aleksandra Seweryn), K.L.-J., M.G.; writing—review and editing,
A.S. (Agnieszka Smieszek), A.S. (Aleksandra Seweryn), K.L.-J., M.G.; visualisation, A.S. (Agnieszka Smieszek),
A.S. (Aleksandra Seweryn); supervision, A.S. (Agnieszka Smieszek), K.Marycz, M.G.; project administration, A.S.
(Agnieszka Smieszek), A.S. (Aleksandra Seweryn), M.G., K.Marycz; funding acquisition, M.G., K.M. (Krzysztof
Marycz). All authors have read and agreed to the published version of the manuscript.
Funding: The financial support from the National Science Centre over the course of the Harmonia 10 project,
entitled ‘New, two-stage scaffolds based on calcium nanoapatite (nHAP) incorporated with iron nano-oxides
(Fe2 O3 /Fe3 O4 ) with the function of controlled release of miRNA in a static magnetic field for the regeneration of
bone fractures in osteoporotic patients’ (Grant No. UMO 2017/26/M/NZ5/01184) is gratefully acknowledged.
Acknowledgments: We are grateful to Ariadna Pielok for helping with qPCR measurements and to Aleksandra
Wierzbicka for support with XRR measurements.
Conflicts of Interest: The authors report no conflicts of interest in this work.

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