Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

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Advanced Industrial and Engineering Polymer Research

journal homepage: http://www.keaipublishing.com/aiepr

In vitro osteoconductivity of PMMA-Y2O3 composite resins

Taigi Honma a, **, Elia Marin a, b, c, *, Francesco Boschetto a, b, d,
Muhammad Daniel bin Idrus a, Kai Mizuno a, Nao Miyamoto b, Tetsuya Adachi b,
Toshiro Yamamoto b, Narisato Kanamura b, Wenling Zhu a, Giuseppe Pezzotti a, b, d, e
a
Ceramic Physics Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki, 606-8585 Kyoto, Japan
b
Department of Dental Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
c
Polytechnic Department of Engineering and Architecture, University of Udine, 33100, Udine, Italy
d
Department of Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, 602-8566, Japan
e
The Center for Advanced Medical Engineering and Informatics, Osaka University, Yamadaoka, Suita, 565-0871, Osaka, Japan

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

Article history: Polymethyl methacrylate (PMMA) bone cements are used as “glue” between orthopedic/orthodontic
Received 4 June 2022 implants and bone, since they can be modeled and easily applied by the surgeon, while being chemically
Received in revised form and biologically stable for many years after surgery. In this research, Y2O3 powder was added to com-
1 August 2022
mercial PMMA bone cement to produce a composite resin, which was then characterized and tested
Accepted 14 August 2022
in vitro to evaluate the cell proliferation and the quality of osteoblastic bone formed in vitro on the
composite. Biological assays showed an increase in cell proliferation on the Y2O3-PMMA composite as
Keywords:
compared to the pristine sample. Alizarin Red staining (ARS) showed the amount of bone formed on a
Osteoconductivity
Composites
composite PMMA resin was about 30% higher than that on the pristine PMMA bone cement reference.
Polymethyl methacrylate The quality of bone tissue was evaluated using Raman spectroscopy, showing the bone tissue formed on
Y2O3 the composite had a better degree of mineralization and a higher maturity as compared to the tissue
Bone grown on the control sample. These preliminary results suggest that Y2O3 plays a biologically active role
in bone growth and Y2O3-composites are affordable, superior candidates as bone cement materials.
© 2022 Kingfa Scientific and Technological Co. Ltd. Publishing services by Elsevier B.V. on behalf of KeAi
Communications Co. Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4.0/).

1. Introduction ability to withstand mechanical stress even in chemically aggres-

PMMA is an amorphous thermoplastic polymer produced from
methyl methacrylate (MMA) monomers and belongs to the family
of acrylic resins. It is used in various applications for it is easy to dye
and work with, in addition to its thermoplastic properties. For
example, it is commonly used as material for optical devices
components such as lens for optical scope or light-guiding plates
for cellphones.
PMMA is also a bio-compatible (bio-inert) polymer commonly
used in the fields of clinical orthopedics and orthodontics. In or-
thopedics, PMMA resins are usually referred to as “bone cement”
and used for fixing implants to bone or repairing bone fractures.
The main reason for choosing PMMA for these applications is its
* Corresponding author. Ceramic Physics Laboratory, Kyoto Institute of Technol-
ogy, Sakyo-ku, Matsugasaki, 606-8585, Kyoto, Japan.elia-marin@kit.ac.jp
** Corresponding author.
E-mail address: elia-marin@kit.ac.jp (E. Marin).
sive environments such as when in contact with biological fluids
and tissues [1]. In the orthodontic field, PMMA resins are used to fill
cavities in the teeth enamel and dentin or to produce dental plates
[2].
Even if PMMA possesses durability and stability, it does not
possess the ability to stimulate cell proliferation or bone formation.
Improving osteoconductivity could drastically reduce the duration
of healing and hospitalization, especially for patients with long-
term bone related diseases such as osteoporosis or osteomalacia
[3,4]. Even in the absence of bone diseases, implant fixation might
fail over time if the implant does not bond well with the bones [5].
Moreover, bacteria can proliferate in the gaps between bone and
implants, resulting in post-operative infections [6].
Bioactive PMMA composite might help to strengthen implant
fixation, stimulate bone regeneration and prevent bacterial adhe-
sion. For these reasons, modern commercial bone cements nowa-
days contain calcium ions to improve bioactivity or antibiotics to
fight against post-operative infections caused by bacteria [7], but

https://doi.org/10.1016/j.aiepr.2022.08.003
2542-5048/© 2022 Kingfa Scientific and Technological Co. Ltd. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: T. Honma, E. Marin, F. Boschetto et al., In vitro osteoconductivity of PMMA-Y2O3 composite resins, Advanced Industrial
and Engineering Polymer Research, https://doi.org/10.1016/j.aiepr.2022.08.003
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

the widespread use of antibiotics in both animals and humans has

contributed to the rise of so-called “superbugs”, namely, bacteria
that have grown resistant to a wide range of antibiotics [8].
There are two main methods for fixation of orthopedic implants,
either with or without the use of bone cements. For example,
cementless HIP joint prostheses are designed with small grooves
that increase the surface area, providing grip for the bone to adhere
[9]. In this kind of fixation, the surface properties such as macro-
and micro-porosity are important factors to consider [10], but the
adhesion can also be enhanced by using bioactive coatings such as
hydroxyapatite or bio-glass [11,12]. The second method of fixation
is by using the acrylic resins as bone cements, which can also
benefit from the presence of bioactive substances in their formu-
lation [13].
Over the years, many additives were used in order to make
PMMA bio-active. A few recent examples being fluorapatite and
graphene oxide, which improved mechanical properties, increased
cell viability and improved cellular adhesion [14], carbon nano-
tubes and graphene oxide, which increased the overall cellular
activity [15]. Even if other innovative materials are often tested, the
most common additives used in PMMA bone cements to improve
their bioactivity are based on calcium-phosphates [16e18], due to
their superior chemical affinity to the surrounding bone tissue.
Y2O3 is commonly used for phase stabilization in zirconia-based
bio-ceramics, referred to as Yttria-Stabilized-Zirconia (YSZ).
Tetragonal zirconia doped with 3 mol.% of yttria is widely used in
the field of restorative dentistry, but similar compositions have also
been applied in the orthopedic field to produce femoral heads and
knee femoral components [19].
Although Y2O3 has been widely used for applications inside the
human body, few investigations have been performed to assess its Fig. 1. (a) Pristine PMMA substrate and (b) composite containing 4.0 vol % of Y2O3. (c)
effect on mesenchymal stem cell lines and their osteoconductivity. Aggregation of Y2O3 particle observed by SEM under 5000  magnification.
Some research indicated that Y2O3 particles possess a strong anti-
oxidant effect that reduces oxidative stress in cells [20].
In this work, after verifying the effects of Y2O3 to the surface biological testing, 4.0 vol % PMMA- Y2O3 composite was chosen as it
morphology and mechanical properties of the composite, the possessed similar mechanical strength to the pristine PMMA
in vitro effects of the Y2O3-PMMA bone cement composite was sample.
investigated. It was found that the addition of Y2O3 has a positive Before all in vitro testing, samples were rinsed with distilled
effect on cell proliferation and bone tissue formation on the com- water and sterilized under UV.
posite according to ARS, SEM/EDS (Scanning Electron Microscopy/
Energy Dispersive X-ray spectroscopy) and Raman imaging. The
2.2. X-ray diffraction (XRD)
Y2O3-added composite improves both quantity and quality of the
bone formed. Considering the results, Y2O3-added composite may
XRD analyses for Y2O3 powder were conducted using a desktop
have a potential for application in the medical field.
MiniFlex300/600 diffractometer (Rigaku, Kyoto, Japan) equipped
with a Cu source, in a 2q/q configuration. The 2q range was
2. Experimental procedure
comprised between 15
and 70
with a step of 0.02
. Measurements
were performed 3 times (N ¼ 3). The obtained data was analyzed by
2.1. Sample production
commercially available software (PDXL. Rigaku, Kyoto, Japan). The
wavenumber of the source was 1.54 Å. The lattice constant was
Commercially available Y2O3 ceramic powder (Shin-Etsu
obtained following the Bragg's law:
Chemical, Tokyo, Japan) was utilized as an additive.
In a preliminary biocompatibility comparison test, pure Y2O3 2dsinq ¼ l (1)
and Si3N4 powders, as well as a 50%/50% mixture of the two, were
molded into green ceramic discs at a pressure 20 KPa and subse- qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
quently tested in vitro. Si3N4 was used as a reference material due a ¼ d h2 þ k2 þ l2 ð AÞ (2)
to the recently increased scientific interest on its potential appli-
cations as a bioactive material. (d: lattice spacing, q: peak position/2, l ¼ 1.54 Å, a: lattice constant,
For composite bone cements, PMMA powder and MMA liquid (h k l): Miller indices).
were provided by a commercial producer (Shofu quick resin O, As an additional analysis for the powder, grain size was calcu-
Shofu, Kyoto, Japan). Four different types of samples were pre- lated following the Scherrer's formula:
pared: one pristine PMMA sample as a control, and PMMA com-
posites containing 1.2 vol %, 4.0 vol %, and 7.3 vol % of Y2O3 powder D ¼ K l=Bcosq ð
AÞ (3)
respectively. The diameter of the aggregated Y2O3 particle was
approximately 1e2 mm (Fig. 1c). The disc diameter and thickness (D: grain size, K: Scherrer's constant (¼ 0.89), B: FWHM).
were about 13 mm and 3.5 mm, respectively (Fig. 1a and b). For The strongest peak (2 2 2) was used for both calculations.
2
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

Fig. 2. Dumbbell shaped tensile testing sample.

2.3. Laser microscopy

Micrographs were taken using 3D laser-scanning microscopy

(VK-X series, Keyence Osaka, Japan). This technique was used to
characterize the surface before the cell culture and to measure the
area covered by bone tissue after the cell culture. The analysis was
performed with micrographs taken under the magnifications of
10  , 20  , and 50  . The microscope used an automated x-y stage
and an autofocus function for z range. The surface roughness values
were obtained from the average of 10 measurements. To evaluate
the influence of Y2O3 powders at a micrometric scale, areas of
50  50 mm were cut from the images acquired at 50  and flat-
tened by using 2nd order baseline removals before measuring.

2.4. X-ray photoelectron spectroscopy (XPS)

Tests were performed using a photoelectron spectrometer (JPS-

9010 MC, JEOL, Tokyo, Japan) with an X-ray source of mono-
chromatic MgKa (output 10 kV, 10 mA). The measurements were
conducted in a vacuum chamber at around 1.0  106, upon setting
Fig. 3. Averaged XRD spectrum of Y2O3 powder. (N ¼ 3).
the analyzer pass energy to 10 eV and the voltage step size to 0.1 eV.

Fig. 4. surface morphology of preliminary green ceramic discs before and after in vitro testing with KUSA-A1 cells; pristine (a) Y2O3, (b) 50%/50% and (c) Si3N4 samples and in vitro
tested (d) Y2O3, (e) 50%/50% and (f) Si3N4 samples, same magnifications.

3
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

Fig. 5. SEM/EDS map of the surface of the sample consisting of 50% Y2O3 powder and 50% of Si3N4 powder, after biological testing in vitro with KUSA-A1.

Fig. 6. Laser micrographs of (a) pristine PMMA sample surface, (b) 1.2 vol% Y2O3, (c) 4.0 vol% Y2O3 and (d) 7.3 vol% Y2O3 composites observed at a magnification of 50  .

4
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

(LabSpec, Horiba/Jobin-Yvon, Kyoto, Japan). All spectra have been

post processed by removing the baseline and reducing the noise
with a moving average filter. After fitting with Gaussian curves, the
intensity, the full width at half maximum (FWHM), and the area of
specific bands (at a range of 1640e1690 cm1 for collagen amide I,
1070 cm1 for carbonate apatite and 960 cm1 for phosphate
apatite) have been used to evaluate the quality of bone tissue, by
calculating the mineral to matrix ratio (RM/M), crystallinity of
apatite, carbonate to apatite ratio and the ratio between hydroxy-
apatite (HAp) and b-tricalcium phosphate apatite (b-TCP).

2.7. Mechanical testing

The tensile testing was performed with dumbbell shaped sam-

Fig. 7. Surface roughness (Ra) of each sample with different concentration of Y2O3 as
measured by Laser Microscopy, red for macroscopic roughness (measured on the
whole 50  images) and blue for the microscopic roughness (acquired on insets of
100 mm  100 mm after flattening).
ples (Fig. 2) using the MCT 2150 Desktop Tensile-Compression
Tester (AND Discover Precision, Tokyo, Japan) at an elongation
rate of 10 mm/min. Tests were repeated three times for each Y2O3
concentration. The Young's modulus was calculated from the initial
slope of the stress-strain curve, while the maximum stress was
obtained directly from the curves.
Compressive testing was performed on cylindrical samples
(length 10 mm, diameter 4 mm) at a strain rate of 1.3 mm/min
following the standard ASTM F451-21.

2.8. Vickers indentation

Table 1
Atomic percentage on the surface of pristine sample and composites, as measured by
XPS. The samples were indented with a Micro Vickers hardness tester
(Micro Hardness tester, SHIMADZU, Kyoto, Japan). Five measure-
Sample Carbon [at. %] Oxygen [at. %] Yttrium [at. %]
ments with different points were taken for each concentration.
PMMA 82.3 ± 1.4 17.7 ± 1.4 N/D Indentations were then observed with Laser microscope and the
PMMA þ1.2 vol% Y2O3 81.0 ± 1.3 18.2 ± 1.2 0.8 ± 0.1
lengths of two diagonals (d1 and d2) from one indentation square
PMMA þ4.0 vol% Y2O3 79.5 ± 1.0 18.9 ± 0.7 1.6 ± 0.5
PMMA þ7.3 vol% Y2O3 78.6 ± 1.4 19.1 ± 1.5 2.4 ± 0.7 were measured, and then, Vickers hardness (HV) was calculated
with following the formula:
a
For both samples, a narrow scan was carried out acquiring 30 scans F ½kgf  2F,sin ½kgf  1:854 h . i
HV ¼   ¼  2 h2 i ¼  2 kgf mm
2
(4)
repetitions on each investigated region and specifically: Carbon S mm 2
(C1s, ~285 eV), Oxygen (O1s, ~530 eV), Yttrium (Y3d, ~160 eV). The
d mm2 d
X-ray angle of incidence and the takeoff angle were 34
and 90
,

respectively. Measurements were performed on 3 different areas d1 þ d2
for each sample (N ¼ 3). The penetration depth of the XPS probe F ¼ 1; d ¼ ; a ¼ 136

2
was in the order of 10 nm.
2.9. Biological testing
2.5. Scanning electron microscopy and Energy Dispersive X-ray
spectroscopy The KUSA-A1 mouse osteoblastic cell line (JCRB, Osaka, Japan)
was selected to form the bone tissue on the samples. The cells were
A field-emission-gun scanning electron microscope (FE-SEM) cultured and incubated in an osteoblast-inducer medium consist-
equipped with an Energy Dispersive X-ray spectroscopy (EDS) (JSM ing of 4.5 g/L of glucose Dulbecco's modified Eagle medium
7001F Scanning Electron Microscope, JEOL, Tokyo, Japan) was used (DMEM) (D-glucose, L-glutamine, phenol red, and sodium pyruvate)
to acquire high-resolution images and chemical composition maps supplemented with 10% of fetal bovine serum. Cells were allowed
of the surface, before and after the cell culture. All images were to proliferate within a Petri dish for 24 h at 37
C with the initial
collected at an acceleration voltage of 15 kV and magnifications of concentration of KUSA-A1 at 5  105 cell/mL. Then, the cultured
100  and 500  before the culture, and of 200  and 1000  after cells were deposited to the surface of the samples. Bone formation
the culture. All samples were sputter-coated (Gressington, Watford, took place within the osteogenic medium, which consisted of
UK) with a thin (20e30 Å) layer of platinum to improve their DMEM supplemented with the following compounds:
electrical conductivity. 50 mg/mL of ascorbic acid, 10  103 M of b-glycerol phosphate,
100  103 M of hydrocortisone, and 10% of fetal bovine calf serum.
2.6. Raman spectroscopy All samples were incubated for 14 days at 37
C while the medium
was changed 4 times during the incubation term.
Raman images and spectra of the sample surface and the bone
tissue produced by KUSA-A1 cells were collected at room temper- 2.10. Viability assay
ature by Raman-touch (Nanophoton, Osaka, Japan) with a
20  optical lens for surface characterization. The excitation source The number of living cells on the surface of the samples after
was a 785 nm laser with a nominal power of 52 mW. The obtained 24 h of the cell culture was analyzed with colorimetric assay based
spectra were analyzed by commercially available software on water -soluble tetrazolium (Cell Counting Kit-8, Dojindo,
5
T. Honma, E. Marin, F. Boschetto et al.
Fig. 8. Raman spectrum of (a) Y2O3 particles, (b) PMMA-4.0 vol% Y2O3 composite and (c) pristine PMMA reference. Raman imaging of (d) pristine reference and (b) Y2O3 particles,
(b) PMMA-4.0 vol% Y2O3 composite. Red signal for Y2O3 (band at 378 cm1), green signal for PMMA (band at 1450 cm1).
Kumamoto, Japan). A water-soluble formazan dye is produced upon
the reduction of the colorimetric indicator (WST-8) induced by the
presence of electron mediator. The amount of the formazan dye
generated indicates the number of living microorganisms present.
Solution was analyzed using microplate readers (Emax, Molecular
Devices, Sunnyvale, California, USA) by collecting the related OD
value.
2.11. Alizarin Red staining
The area where bone formed was visualized by Alizarin Red
staining (ARS) (Sigma Aldrich, Tokyo, Japan). The cells were rinsed
with distilled water twice and then fixed with 4% Para-
formaldehyde phosphate buffer solution (PFA) (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). The bone tissue was left
stained with ARS for 30 min at room temperature. After stained, the
samples were rinsed 5 times with distilled water to remove excess
ARS. Then, micrographs of the surface were taken with Laser mi-
croscope under magnification of 10  and 20  . For the quantifi-
cation of staining density, the staining was removed with 10%
Formic acid (Wako Pure Chemical Industries, Osaka, Japan) and the
concentration was determined by measuring the absorbance at
450 nm.
2.12. Statistical analysis
All the data were expressed as mean values ± one standard
deviation and were analyzed for their statistical significance using
ANOVA multi-variance test; p values which smaller than 0.01 and
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
0.05 are considered statistically relevant with two asterisks (**)
and statistically relevant with one asterisk (*) respectively. Values
of p larger than 0.05 were considered as statistically non-significant
and marked as “n.s.”.
3. Results
3.1. Y2O3 powder characterization
Fig. 3 shows the average XRD diffraction pattern as obtained on
the ceramic reinforcement powder. Peaks associated with Y2O3 can
be observed around 2q ¼ 21
, 29
, 34
. 48
and 58
. The composi-
tion of this powder determined with PDXL software based on the
peak position was (Y 0.95: Eu 0.05)2O3. Moreover, the crystal
structure was determined to be cubic by the analytical software. In
result of calculation using peak at 29.2
(2 2 2), lattice constant was
10.57 ± 0.01 Å and, the lattice volume was 1.18 nm3. Grain size
computed using the (2 2 2) peak followed the Scherrer's formula
and was determined to be 33.24 ± 1.02 nm.
3.2. Preliminary in vitro comparison with Si3N4
Fig. 4 shows the surface morphologies of the preliminary green
ceramic discs before and after in vitro testing with KUSA-A1
mesenchymal cells. On all three compositions, 100% Y2O3, 50%
Y2O3/50% Si3N4 and 100% Si3N4, deposits of mineralized tissue were
formed, but while for Si3N4 the deposits resulted in a thin layer
covering the ceramic crystals, the samples containing Y2O3 showed
large aggregates with complex, three dimensional morphologies,
T. Honma, E. Marin, F. Boschetto et al.
Fig. 9. Scanning electron microscope (SEM) images of (a) pristine PMMA sample surface, (b) 1.2 vol% Y2O3, (c) 4.0 vol% Y2O3 and (d) 7.3 vol% Y2O3.
Fig. 10. EDXS analysis results for the pristine PMMA polymer and the composites with
different fractions of particulate.
suggesting that the presence of Y2O3 leaded to the formation of
much higher amounts of mineralized tissues.
Fig. 5 shows the morphology of the surface of the sample con-
taining both ceramic powders after in vitro testing with KUSA-A1.
The optical imaging has been combined with the SEM/EDS maps
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
in order to identify the locations of the three main elements: sili-
con, yttrium and calcium. Carbon, another major constituent of the
EDS maps and mainly associated with the organic materials, is not
shown in Fig. 5 due to its presence at all locations while phos-
phorous, the second main constituent of the bone apatite, is not
shown as it overlaps perfectly with calcium. Even if a weak carbon
signal was measured on the whole map, it can be observed that the
mineralized tissue is preferentially formed on the surface of Y2O3
particles, where it grows to form large deposits. This result suggests
that Y2O3 might be a more effective reinforcement ceramic for bone
cements when compared to Si3N4.
Fig. 6 shows the pristine morphology of the surface of the
pristine PMMA sample (Fig. 6a) and the PMMA-Y2O3 composite
(Fig. 6b). Fig. 6c shows the surface roughness (Ra) value measured
from the different samples. Both samples showed the characteris-
tics spherical bubbles, which were formed during the polymeri-
zation reaction of PMMA resins. The heat from the exothermic
reaction caused the dissolved air inside the MMA liquid to be
released thus forming the bubbles. These bubbles have an average
diameter of 50 mm. The particles of Y2O3 are clearly visible as a
white, well-dispersed phased on the surface of the composite
material (Fig. 6b, c and d), in particular in comparison with the
smooth surface (Fig. 6a) of the pristine control. The presence of the
particles resulted in a slight change in the morphology of the sur-
face, which appeared more wrinkled. The particle dispersion in the
composite was observed to be homogeneous at all concentrations,
when observed at relatively low magnifications.
As shown in Fig. 7, there were no significant differences in
macroscopic surface roughness between the four types of samples,
proving that the addition of Y2O3 particles did not influence the
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

Fig. 11. (a) Stress-strain curve measured by tensile test, (b) Stress of each different concentration of PMMA-Y2O3 composites, and (c) Young's modulus of each sample.

Table 2 near-infrared laser as the excitation source. In the spectrum of the

Ultimate compressive strength for the different samples. Y2O3 particles, shown in Fig. 8a, some relatively intense bands are
Sample Ultimate strength [MPa] Modulus [GPa] identified as good indicators for the presence of Y2O3 in the com-
posites. A total of 9 bands could be identified and in particular two
PMMA 86.4 ± 5.3 3.4 ± 0.4
PMMA þ 1.2 vol% Y2O3 88.1 ± 3.4 4.1 ± 0.4 bands at relatively low Raman shifts, 378 and 470 cm1 (Fg þ Ag).
PMMA þ 4.0 vol% Y2O3 91.4 ± 4.2 4.6 ± 0.6 Bands at higher shifts, 1334, 1363, 1535, 1563, 1598, 1799 and
PMMA þ 7.3 vol% Y2O3 84.6 ± 11.5 5.1 ± 0.8 1981 cm1 are not actually Raman bands but infrared photo-
luminescence bands caused by the presence of small amounts of
contaminants, in particular rare earth elements [21].
overall morphology of the surfaces. When the roughness is For PMMA, the bands appearing in Fig. 8c are caused by
measured on a micrometric-scale, however, the presence and vs(CeCeO), at 601 cm1, vs(CeOeC) at 815 cm1, a-CH3 rocking at
amount of reinforcing particulate plays a clear role, as shown by the 970 cm1, OeCH3 rocking at 990 cm1, ds(CeH) of OeCH3 at
blue bars. A statistically relevant increase in micro-surface roughness 1450 cm1 and finally C]O symmetric stretching at 1730 cm1
is shown between each increment of the amount of particulate. [22].
Table 1 shows the atomic percentage measured by XPS at the The mixture of PMMA and Y2O3, here represented in Fig. 8b by
external surface of each sample. Despite the low reliability of XPS the average spectrum collected on the sample containing 4% vol. of
data when focusing on carbon contents (carbon signals are usually Y2O3 particulate, clearly shows the presence of both molecules. It
overestimated due to surface contamination), it can be observed can be observed that most of the bands maintain the original in-
that the amounts of both oxygen and yttrium increase with the tensity ratio of the pristine spectra (cf. either Fig. 8a or Fig. 8c) with
amount of particulate, confirming that a fraction of the particles is the important exception of the PMMA band located at 990 cm1
exposed. The amount of exposed particles seems to increase with and assigned to OeCH3 rocking. This results indicate that the
the concentration in a linear fashion, suggesting that high contents presence of Y2O3 during the setting of the PMMA resin lead to a
of ceramic reinforcement could potentially lead to more bioactive higher oxidation of the polymer, probably due to the interfering of
composites. the particulate with the polymerization process.
Fig. 8 shows the most significant results of the Raman spec- Fig. 8d and e shows the Raman imaging results on the pristine
troscopy and imaging experiments, as collected using a 785 nm PMMA reference sample (green) and the composite containing 4%

8
T. Honma, E. Marin, F. Boschetto et al.
Fig. 12. (a) Laser micrographs of the indentation and (b) Vickers Hardness (HV) of each type of samples.
Fig. 13. Optical density (OD) of the WST-8 viability assay for living KUSA-A1 cells
grown on the different samples. (N ¼ 5).
vol. of Y2O3 particulate. It can be observed that, at these magnifi-
cations, the distribution of Y2O3 (red) seems to be more or less
homogeneous on the surface of the composite sample, with the
exception of the polymeric bubbles formed on PMMA due to the
presence of residual gas, where the signal of Y2O3 appears to be
weaker. These results are in line with the previous images acquired
by laser microscopy, where the Y2O3 particulate appeared to be
homogeneously distributed on the surface of the samples.
Fig. 9 shows SEM maps of the surface of pristine PMMA sample
and the various PMMA-Y2O3 composites, going from 1.2 vol% (b),
4.0 vol% (c) and finally 7.3 vol% (d). The relative atomic percentage
of elements measured by EDS is shown in Fig. 10. In Fig. 6b, the
presence of Y2O3 particles can be observed mainly as a roughening
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
of the otherwise smooth surface of the pristine PMMA (cf. Fig. 9a).
As the fraction of ceramic particulate is increased, the secondary
phase seems to start protruding from the polymeric matrix, con-
firming a progressive increase in the roughness of the surface.
Fig. 10 shows the average chemical composition of the four
types of samples, as a function of particulate fraction. It can be
observed that all materials appear to be highly oxidized, with ox-
ygen content between 16% and 20%. This is probably due to the low
reliability of EDXS for the concentration of very light elements like
carbon and oxygen. Nevertheless, the yttrium concentration in-
crease with the fraction of particulate, further supporting the hy-
pothesis that the powders are well dispersed, at least in the first
few microns below the surface which account for the depth of the
EDXS probe.
3.3. Mechanical properties
Fig. 11 shows the results of tensile tests of the different samples.
Fig. 11a shows the representative stress-strain curves for four
samples containing different fractions of yttria. The strength and
Young's modulus were calculated from stress-strain curve and
shown in the histograms (Fig. 11a and b). In Fig. 11b, the pristine
PMMA sample has the highest strength, while strength decreased
by about 25% for all the composites regardless of the concentration
of Y2O3. Furthermore, the more the ceramic powder added, the
larger the data scattering. This was probably caused by the
decreasing homogeneity of PMMA due to the addition of Y2O3
powder. Young's modulus was calculated from each stress-strain
curve (Fig. 11c). For Young's modulus, pristine PMMA sample
showed the highest value. From Fig. 11c, we can deduce that as the
amount of Y2O3 powder increases, the stiffness of the composite
decreases. On the other hand, from the different composites, 4.0 vol
% PMMA-Y2O3 composite had the most similar value of Young's
modulus compared to the pristine PMMA sample. Other than ten-
sile, compressive stresses were also measured following the stan-
dard ASTM F451-21. Results, summarized in Table 2, show that the
T. Honma, E. Marin, F. Boschetto et al.
Fig. 14. Images of (a) pristine PMMA sample and (b) PMMA- 4.0 vol% of Y2O3 composite stained with ARS. Laser micrographs of the stained surface of (c), (e) pristine PMMA sample
and (d), (f) PMMA- 4.0 vol% of Y2O3 composite under magnification of 10  and 20  respectively.
ultimate strength of the composite and, in particular, the elastic
modulus increase with the fraction of particulate up to the highest
concentration, where despite the high elastic modulus the ultimate
strength drops.
Fig. 12 shows the results of the indentation testing performed on
the surface of the samples. Fig. 12a shows the Vickers hardness ob-
tained from the images and the formula in (4), expressed in HV
(GPa), as a function of the fraction of Y2O3 particulate. The measured
hardness seems to be more or less constant, with only the high
fraction composite showing a different behavior and, in particular, an
higher average hardness but with a larger distribution.
3D images of the hardness indentations from the reference
PMMA and the samples containing 1.2 vol% and 4.0 vol% of Y2O3
confirm, shown in Fig. 12 b, c and d, further support the observation
that the hardness of the three samples is similar, as the tree prints
have comparable diagonal lengths. For the sample containing
7.3 vol% Y2O3, the hardness seems to greatly fluctuate depending on
the location. This is supported by the indentations in Fig. 12e and f,
one of which is much smaller than the other.
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3.4. In vitro osteoconductivity test
Fig. 13 shows results of optical density measured from the
viability assay. The optical density is proportional to the number of
living cells present on the surface of the sample. The composites
showed higher optical densities when compared to the pristine
PMMA sample and the negative control. The composite containing
4.0 vol% of Y2O3, showed a 25% increase in optical density, which
can be translated as a 100% increase in the number of cells,
considering the value of the negative control that only contains PBS
solution the other two composites show comparable behaviors,
with a larger statistical dispersion for the composite containing
lower fractions of particulate.
Fig. 14 shows images of the pristine PMMA sample and 4.0 vol%
of Y2O3 composite after the samples were stained with ARS [23].
The stained areas appeared to be wider on surface of the composite
than that of pristine PMMA sample. In both cases, the staining did
not cover the surfaces of the samples completely and preferential
distributions can be clearly observed in concave areas made by
T. Honma, E. Marin, F. Boschetto et al.
Fig. 15. Optical density (OD) value of the Alizarin red stains (ARS) for each sample.
scratches and small pores. By comparing Fig. 14e and f, it can be
observed that, for the composite, the staining also spread outside of
the porous area. The presence of calcium ions was measured by ARS
staining [24] and the results showed that cells with apatite covered
larger portions of the PMMA-Y2O3 composite compared to the
Fig. 16. Raman imaging maps collected on the surfaces of the (a) PMMA reference sample and (b) composite sample containing 4 vol% of Y2O3 (Green: HAp, Red: PMMA, and Blue:
Y2O3). Normalized Raman spectra after in vitro testing for both samples in the spectra regions between (a) 900 cm1 to 1225 cm1 and (b) 1400 cm1 to 1850 cm1.
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
pristine PMMA sample. When compared to the sample containing
4.0 vol% Y2O3, the other two composites showed comparable re-
sults (not shown in Fig. 14).
On composite resins, the OD value of ARS (Fig. 15) was between
5 and 40% higher respect to the pristine PMMA reference. As the OD
value is related to the amount of Ca ion which is produced by cells
on the surface, the KUSA-A1 cells on the composite showed more
active osteogenic reaction compared to the pristine PMMA sample.
Fig. 16 shows the Raman spectra and imaging of the surface of
the reference PMMA sample and the sample containing 4 vol% of
Y2O3 after in vitro testing with KUSA-A1 cells. The spectra, collected
in the two regions marked in yellow, were normalized on the main
peak of PMMA (1450 cm1). The regions were divided in two areas:
from 900 cm1 to 1225 cm1 and from 1400 cm1 to 1850 cm1. In
the first region (Fig. 16c), the composite and the pristine PMMA
spectra are dominated by the peak related to phosphate stretching
1
vibration (n1 PO3
4 ), located at 960 cm . Additionally, two shoulder
bands at 946 cm1 and 970 cm1 were observed and identified as
carbonate aptatite and tricalcium phosphate (TCP), respectively.
Two additional peaks at 1030 cm1 and 1070 cm1 were caused by
phosphate asymmetric stretch and the b-type carbonate symmetric
stretch. In the second region, the dominant peak was PMMA
(1450 cm1, CH2 stretching vibration) and a relatively weak peak at
1660 cm1 which was associated with Amide I as a marker of
collagen. The Raman imaging shows the intensity of the apatite
mineral with peak at 960 cm1 (phosphate stretching vibration)
labelled as green tones, and of PMMA with the peak at 1450 cm1 as
red tones. On the other hand, the intensity of Y2O3 is presented as
peaks at 1535 cm1 and 1563 cm1 was labelled in blue color.
T. Honma, E. Marin, F. Boschetto et al.
Fig. 17. Bone quality as evaluated by using Raman spectroscopy, (a) Quality of bone formation on each surface, FWHM of 960 cm1 band, (b) Carbonate-to-phosphate ratio, (c) HAp-
to-TCP ratio, (d) Mineral-to-matrix ratio. (n ¼ 8).
Fig. 12e shows the overlapping of PO3 4 phosphate stretching vi-
bration band attributing to HAp (green) at 960 cm1 and CH2
stretching vibration band of PMMA (red) at 1450 cm1. Form the
image it proved that the composite sample was covered with a
larger amount of bone formation than the pristine sample.
Fig. 17a shows the Full width at half maximum (FWHM) of the
960 cm1, phosphate PO3 4 band which is related to mineral hy-
droxyapatite. Mineral crystallinity can be estimated using FWHM: a
narrow FWHM of the phosphate band (960 cm1) represents a
higher degree of crystallinity. The FWHM which was observed on
the composite was narrower compared to that on the pristine
sample. This indicates that the bone produced on the composite
reached higher bone crystallinity, which is also a considered
marker for good development and overall health of bone [28].
Fig. 17b shows the carbonate-to-phosphate ratio which in-
dicates carbonate ion substitution in the apatite mineral, calculated
with the ratio of the area of carbonate apatite band (1070 cm1) and
the area of phosphate band (960 cm1). This parameter shows
significant changes with different samples. Bone produced on the
PMMA-Y2O3 composite had smaller carbonate-to-phosphate ratio
than the pristine PMMA sample [30].
Fig. 17c shows the ratio of area of the main HAp band (960 cm1)
and the sum of area of b-TCP bands (946 cm1 and 970 cm1).
Results showed the amount of HAp on the composite was higher
than the control sample.
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
Fig. 17d shows the result of mineral-to-matrix ratio (RM/M),
which provides information of the composition of the bone tissue.
This value was obtained from the ratio of area of the peak of
phosphate band at 960 cm1 and the peak at 1660 cm1 (related to
the Amide I vibration in collagen). RM/M value indicated there were
no significant differences in the level of mineralization of bone
formations on both samples [31].
Fig. 18 show SEM images of the surface of pristine PMMA sample
and PMMA-Y2O3 composite after culture of KUSA-A1. In Fig. 18a and
c, it was observed that a layer of bone tissue formed on the pristine
PMMA sample, but the layer appeared to be relatively thin as the
surface of the sample is still clearly visible. On the other hand, the
surface of the composite sample Figs. 18b and d was covered in a
thicker layer of bone tissue, with some localized deposits pro-
truding from the surface for more than 10 mm. As calcium and
phosphorus are the main elements of bone, Fig. 18e shows that
there are much more of these elements on the composite than the
pristine PMMA sample. The amount of carbon of composite is lower
than of the PMMA, means that there is more bone covered on the
surface of PMMA-Y2O3 composite.
Fig. 19 shows the ratio of the EDS signals from calcium and
phosphorous, as extracted from Fig. 18e. As the amount of Y2O3 is
progressively increased, so is the amount of calcium, indicating a
higher maturity of the bone tissue formed, which gets closer to the
theoretical value for healthy bone (Ca/P ¼ 1.6).
T. Honma, E. Marin, F. Boschetto et al. Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx

Fig. 18. SEM images of the bone formation on surface of (a) pristine PMMA sample and (b) PMMA-Y2O3 composite under magnification of 200  . EDS images of the bone on the
surface of (c) pristine PMMA sample and (d) PMMA-Y2O3 composite under magnification of 1000  (Blue: Calcium, Green: Phosphorus, Red: Carbon) (e) Atomic percentage of bone
formation on surface of each sample after cell culture.

4. Discussion without decreasing the bone quality compared to common PMMA

bone cement.
PMMA-Y2O3 composite promoted proliferation of KUSA-A1 cells Analysis based on SEM, EDS and Laser microscopy before cell
and stimulated much larger amounts of bone tissue formation, culture demonstrated that the composite had Y2O3 ceramic powder

13
T. Honma, E. Marin, F. Boschetto et al.
on the outer surface, but these particles did not lead to a significant
increase in surface roughness. One of the main reasons was prob-
ably due the aggregation of the particles as the diameter of the
clusters observed with SEM ranged around 1e2 mm. Suggested by
M. Lampin et al. [32], cell attachment ability should be affected by
the surface roughness. From the results we can conclude that
addition of Y2O3 powder up to at least 7.3 vol% may maintain a
propensity of cell attachment similar to that to pristine PMMA bone
cement, only from the viewpoint of surface roughness.
Analysis for mechanical properties by tensile test and indenta-
tion suggested that the addition of Y2O3 powder into PMMA sample
caused decrease in strength and stiffness even if there were no
significant differences. Additionally, for strength, the increase of
standard deviation in the results shows PMMA lost its homoge-
neities in proportion to the amount of Y2O3 powder added. Simi-
larly, the same pattern was observed for indentation. Especially, the
sample containing 7.3 vol % of Y2O3 powder showed an exceptional
change in mechanical properties compared to the other samples.
The results of viability assay proved that PMMA-Y2O3 composite
promoted better cell proliferation if compared to that with the
pristine PMMA sample as shown in Fig. 9. Y2O3 powder is likely to
affect the metabolism of the cells. Mitochondria in cells produce
reactive oxygen species (ROS) which have negative effects to cells.
Y2O3 are known as free radicals’ scavenger [33] and may act as an
antioxidant, protecting the surrounding cells from the ROS [16]. As
reported by Xiang song et al. [34], Y2O3 particles also contributed to
the inhibition the ROS/NF-k-B pathway. It is possible that Y2O3
particle reduced the oxidative stress of KUSA-A1 cells during the
proliferation thus enable the contribution of higher number of
living cells. The images of the surface and optical density value (OD
value) after the cell culture and in vitro testing indicated that Y2O3
had a positive effect on bone production.
Moreover, RM/M of each sample showed no significant differ-
ences (Fig. 13d), even though Y2O3 promoted the production of
much more bone matrix as shown in Figs. 10e14. This fact indicates
that the amount of type I collagen which works as a scaffold for
bone formation [35e37] existed plentifully on the composite
compared to the pristine PMMA control sample. Based on biological
results, it can be concluded that Y2O3 has an ability to create much
better environment for cell by reducing ROS, and then these cells
could produce collagen proportionally thus making it possible to
increase the amount of bone tissue formed on the scaffold.
For bone quality, PMMA-Y2O3 composite showed an ideal RM/M,
a value that indicates the ratio between organic and inorganic
Fig. 19. Ca/P ratio as measured by using EDS on the surface of the different samples.
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Advanced Industrial and Engineering Polymer Research xxx (xxxx) xxx
components in bone structure [31]. This result should support that
the composite does not decrease the relative mineral content and
maintain the “healthy level” of bone tissue. Moreover, based on the
both results of FWHM and carbonate-to-apatite ratio, the bone
crystallinity on the composite increased and the ion substitution
ratio in the HAp lattice decreased (Fig. 13a and b). Decrease in HAp
crystallinity should be induced through ion substitution in the
crystalline lattice [38] and the integration of carbonate ion causes
distortion in the crystal structure of apatite [39e43]. In this work, it
is predicted that the composite may induce cell to produce more
stable apatite especially in terms of solubility in water or acidic
environmental [44]. In addition, the result of the ratio between HAp
and b-TCP (Fig. 13c) indicates that the composite should produce
better quality bone tissue compared to the pristine PMMA sample
because as reported by L. Sun et al. [45], relative biological solubility
of these two substances is: b-TCP > HAp.
In short, PMMA-Y2O3 composite should have contribute to not
only improving both the quantity and the quality of the bone.
5. Conclusion
In this research, bioactive bone cement consisting of PMMA and
Y2O3 was produced by simply mixing a commercially available
PMMA resin with the Y2O3 ceramic powders.
- Results suggest that PMMA-Y2O3 composite has a positive effect
on KUSA-A1 osteoblastic cells as it stimulates cell proliferation
and bone production, while increasing bone tissue quality
compared to common PMMA bone cement;
- From the viewpoint of mechanical properties, addition of Y2O3
powder to PMMA affected its strength, Young's modulus, and
hardness negatively. Moreover, data scattering in those value
was also influenced by the amount of Y2O3 powder added to the
composite;
- PMMA-Y2O3 composite has potential for application in ortho-
pedics and orthodontics.
The authors want to emphasize that this manuscript contains
preliminary characterizations that should be supported with
additional in vitro cytotoxicity experiments, as well as long-term
performances in simulated body fluids, before moving to in vivo
experiments.
CRediT authorship contribution statement
Taigi Honma: Writing-original draft, Formal analysis, Inves-
tigation.
Elia Marin: Conceptualization, Methodology, Project adminis-
tration, Writing-review & editing, Supervision.
Francesco Boschetto: Methodology, Investigation, Supervision.
Muhammad Daniel bin Idrus: Investigation, Writing-review &
editing.
Kai Mizuno: Investigation.
Nao Miyamoto: Resources.
Tetsuya Adachi: Resources, Formal analysis.
Toshiro Yamamoto: Resources, Supervision.
Narisato Kanamura: Resources, Supervision.
Wenling Zhu: Formal analysis, Writing-review & editing,
Supervision.
Giuseppe Pezzotti: Writing-review & editing, Supervision.
Funding
This work was supported by JSPS KAKENHI Grant Number
18K17175.
T. Honma, E. Marin, F. Boschetto et al.
Conflicts of interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: Elia Marin reports financial support was provided by
Japan Society for the Promotion of Science.
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