Prof. dr.

Simona Cavalu
Faculty of Medicine and Pharmacy
University of Oradea
ROMANIA
Motivation
As the average age of population grows, the need for
medical devices/biomaterials to replace damaged or
worn tissues increases.
As patients have become more and more demanding
regarding esthetic and biocompatibility aspects of
their dental/orthopedic restorations .
The field of tissue engineering is highly
interdisciplinary
Brings together people with knowledge in materials
science, biochemistry, cell biology, immunology, and
surgical expertise to solve a range of open problems.
The successful design of tissue-engineered constructs
drives the need to design novel biocompatible
materials and study their interactions with living cells.
Tissue engineering evolved from the field of
biomaterials development and refers to the practice of
combining scaffolds, cells, and biologically active
molecules into functional tissues.
Bioceramics investigated in the
present study
Poly (methylmethacrylate) (PMMA)
bone cements:
are extensively used in certain types
of total hip or total knee
replacements
are of potential utility wherever
mechanical attachments of metal to
living bone is necessary
The main function of the cement is
to serve as interfacial phase between
the high modulus metallic implant
and the bone, thereby assisting to
transfer and distribute loads.
Alumina/zirconia ceramics were
successfully used in total hip/knee
arthroplasty in the last decades.
For dental application: root canal
posts, orthodontic brackets, implant
abutments and all- ceramic
restaurations
is a high performance biocomposite
that combines the excellent material
properties of alumina in terms of
chemical stability and low wear, and
of zirconia with its superior
mechanical strength and fracture
toughness.
PMMA bone cement
Alumina/zirconia bioceramics
Motivation
The surface modification and post-synthesis treatment also influences
the performances of the bioceramics designed to dental and ortopedic
applications.
According to their interaction with surrounding tissue, bioceramics
can be categorized as bioinert or bioactive.
Tough and strong ceramics like zirconia, alumina or alumina-zirconia
composites are not capable of creating a biologically adherent
interface layer with bone due to the chemically inert nature of these
two stable oxides .
PMMA cements cannot adhere to existing bone, but this
disadvantage may not be as pertinent for vertebroplasty as for
arthroplasty, because is injected directly into the bone instead using
as an adhesive agent.
Surface modification: organic coating
The use of surface covering layers (i.e. coatings) provides
methods to control the biological response to materials and
material devices including implants and prostheses.
Several types of organic materials can be used to generate
a coating with specific modulatory effects on the
biological response. Examples include proteins, DNA,
sugars, etc.
Specific biological responses that can be controlled are cell
attachment and behavior.
Organic coatings consisting of proteins are generally based
on the presence of these proteins at the implant location
[S. Cavalu &all, Key Engineering Materials Vol. 583 (2014) pp 101-106]
Surface modification: inorganic molecules
Many different techniques are currently in use to condition the
surfaces of abutments and fixtures of implants: surface blasting
or acid etching can increase the rate and amount of new bone
formation on the implant surface.
The administration of complex fluorides as compared with NaF
suggests the possibility of using them as effective agents in
dental caries prevention in human populations.
For example, stannous fluoride converts the calcium mineral
apatite into fluorapatite, which makes tooth enamel more
resistant to bacteria generated acid attacks.
[F. Hattab, The State of Fluorides in Toothpastes, J. Dent., 17, 4754 (1989)].
Goal
In the present study we are focused on the possible
beneficial effect of PMMA/Ag
2
O collagen coated
respectively and surface modification of alumina /zirconia
bioceramics by fluoride treatment
The surface modifications of alumina/zirconia
bioceramics are investigated upon different treatments
with sodium tetrafluoroborate and stannous fluoride
respectively.
The main objective is to analyze the biocompatibility of
new bone substitute upon surface treatment, via in vitro
and in vivo tests.
Goal
PMMA modified by Ag2O
addition and collagen coating
80%Al2O3- 20%ZrO2 modified
by surface fluoride treatment
Influence on fibroblasts
viability, attachment and
proliferation
Biomaterials: PMMA bone cement
Ag
2
O doped PMMA is proposed as an alternative to
antibiotic loaded cements, silver being capable of killing
over 650 forms of bacteria, viruses .
The antimicrobial efficacy of these composites depends on
their ability to release the silver ions fromthese composites
upon interaction with biological fluids.
It has been previously demonstrated that biomimetic
coatings consisting of collagen type I are suitable surfaces
to enhance their bioactivity, cell attachment and
proliferation [S. Cavalu & all. Digest Journal of Nanomaterials and Biostructures , 2010]
PMMA/Ag
2
O bone cement
As antimicrobial agent, Ag
2
O particles were incorporated in
PMMA with respect to the total powder amount in a
concentration ranging from0.1%to 4 %w/w.
Surface morphology (SEM) of the PMMA/Ag
2
O specimen surface before any
treatment: a) 0.5%Ag
2
O, b) 1%Ag
2
O and c) 2%Ag
2
O.
Kinetics of Ag
+
release from the PMMA specimens with
different silver oxide content, during 21 days incubation
in Simulated Body Fluid
0 5 10 15 20 25
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.10%
0.25%
0.50%
1.00%
2.00%
4.00%
A
g
+

c
o
n
c
e
n
t
r
a
t
i
o
n


(
m
M
)
Time (days)
Possible mechanism of the antimicrobial
action of silver ions :
Is not completely known
Possible interaction with thyol group compounds
found in the respiratory enzymes of the bacterial cells.
Silver binds to the bacterial cell wall and cell
membrane and inhibits the respiration process.
In case of E-coli, silver acts by inhibiting the uptake of
phosphate and releasing phosphate, mannitol,
succinate, proline and glutamine fromthe E-coli cells.
In addition, it was shown that Ag+ ions prevent DNA
replication by binding to the polynucleotide
molecules, hence resulting in bacterial death .
Electrodeposition of soluble collagen type I
3500 3000 2500 2000 1500 1000 500
-0.02
0.00
0.02
0.04
0 2 4 6 8 10
0
2
4
6
8
10
6
4
0
1
1
4
0
1
2
4
0
1
4
3
6
1
7
2
2
2
9
5
0
A
b
s
o
r
b
a
n
c
e
/
A
r
b
i
t
r
a
r
y

u
n
i
t
s
Wavenumber / cm
-1
3
1
8
0
2
9
5
0
1
7
2
2
1
6
3
5
1
5
5
0
1
4
3
6
1
2
4
0
1
1
4
0
1
0
3
5
9
8
5
6
4
0
ATR FTIR spectra recorded on the surfaces of the Ag
2
O/PMMA before and
after collagen electrodeposition. Distinct peaks of collagen: amide I at 1635
cm
-1
(C=O stretching), amide II at 1550 cm
-1
(N-H deformation) and amide
III around 1200 cm
-1
(combined N-H bending and C-N stretching).
ATR FTIR spectrum of native collagen type I (a), deconvoluted amide I native
collagen (b) and adsorbed collagen to PMMA specimens with 0.5% Ag
2
0 (c),
1% Ag
2
0 (d) and 2% Ag
2
0 (e) respectively.
1800 1600 1400 1200 1000 800 600
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
0.225
0.250



1
2
2
8
A
m
i
d
e

I
I
I



Wavenumber cm
-1
A
b
s
o
r
b
a
n
c
e



(
a
.
u
.
)


1
6
4
0
A
m
i
d
e

I


1
5
4
6
A
m
i
d
e

I
I
a)
1600 1620 1640 1660 1680 1700
b)


A
b
s
o
r
b
a
n
c
e



(
a
.
u
.
)
Wavenumber (cm
-1
)
1600 1620 1640 1660 1680 1700
A
b
s
o
r
b
a
n
c
e

(
a
.
u
)
Wavenumber (cm
-1
)
d)
1600 1610 1620 1630 1640 1650 1660 1670 1680 1690
-0.000005
0.000000
0.000005
0.000010
0.000015
0.000020
e)



Wavenumber cm
-1
A
b
s
o
r
b
a
n
c
e



(
a
.
u
.
)
Collagen
amide I
helix helix helix turns
(cm
-1
) A (%) (cm
-1
) A (%) (cm
-1
) A (%) (cm
-1
) A (%)
native
collagen
1630 28.3 1644 33.2 1665 34.7 1682 3.8
Specimen 1
0.5% Ag
2
O
1625 40.2 1641 25.5 1657 23.5 1670 10.8
Specimen 2
1% Ag
2
O
1619 4.2 1637 37.7 1657 43.5 1682 14.6
Specimen 3
2% Ag
2
O
1630 34.0 1640 44.0 1663 12.0 1673 10.0
Characteristics of FTIR bands
Specific components within the fine structure of amide I adsorbed
collagen is correlated with different states of hydrogen bonding
associated with the local conformations of the alpha chain peptide
backbones.
The highest frequency carbonyl absorption peak represents the
weakest H-bonded system .
The peak located in the higher region, at 1682 cm-1, represent the
formation of an antiparallel -sheet structure (or turns).
As a general behavior, one can observe a shift toward lower frequencies,
a decrease in helix total content and concomitant increase of turn
percentage upon adsorption, as a consequence of denaturation.
Surface morphology of the PMMA specimens surface after
collagen electrodeposition (d, e, f) and upon incubation in SBF
during 21 days (g, h, i).
0.5%Ag2O 1%Ag2O 2%Ag2O
The formation of hydroxyapatite crystals was strongly influenced by the
presence of collagen layer, but dependent on the silver oxide concentration as
well. [S. Cavalu& all, 2010]
Morphology of fibroblasts after 24 h incubation with PMMA
specimens. The fibroblasts showed a wide variety of shapes: spread
multipolar or round , as well as spindle shaped, elongated cells
0.5%
1%
2%
Human fibroblasts
(HSFs) in a density
of 2x10
4
cells/cm
3
were seeded upon
each PMMA
specimen substrate
Results shows viable fibroblasts cells with respect to control and PMMA/Ag with
different concentration of silver oxide after 3, 12 and 24 hours of culture (p< 0.05).
Initial cells attachment is influenced by the silver content in the samples.
The results shows a progressive decrease in optical density after 3 hours, with
higher silver concentration. The sample containing 1% silver oxide exhibits
comparable behavior to that of control (commercial cement).
Fibroblasts
viability by MTT
assay
Biomaterials:
Alumina/zirconia ceramic
Composition : 80%Al
2
O
3
20%3YSZ;
Prepared using a spark plasma sintering method
Characterization made by FTIR and XRD spectroscopy
Morphological details of the surface investigated by SEM.
S. Cavalu & all, Int. J. Appl. Ceram. Tech. (2014)
Surface treatment with fluoride
ATR FTIR evidence
Fig. 1 ATR FTIR spectra of SnF2 and
NaBF4 powders as received from the
supplier .
Fig. 2 ATR FTIR spectra recorded on
specimen surface before and after
treatment using SnF2 and NaBF4.
Al-O
Zr-O
Surface treatment with fluoride- XPS
evidence
1200 1000 800 600 400 200 0
F

1
s
A
l

2
s
Z
r

3
d
A
l

2
p
C

1
s
N

1
s
O

1
s
S
n

4
d


Z
r

4
p

F

2
s

S
n

3
p
1
S
n

3
d
Z
r

3
d
N

1
s
F

1
s
A
l

2
p
N
a

1
s
O

1
s
C

1
s


I
n
t
e
n
s
i
t
y

(
a
.
u
)
Binding Energy (eV)
S
n

3
p
3

A
l

2
s
O

A
u
g
e
r
Z
r

4
p
Specimen 2
SnF
2
NaBF
4
Why fluoride?
Administration of complex fluorides suggests the
possibility of using them as effective agents in dental
caries prevention.
Stannous fluoride converts the calcium mineral apatite
into fluorapatite, which makes tooth enamel more
resistant to bacteria generated acid attacks.
NaF has been known to be one of the most effective
agents for the treatment of vertebral osteoporosis by
its stimulating effect on new bone formation.
In vitro test: cells culture
Human fibroblast (HLF) seeded in a concentration of 2x10
4
/cm
2
cells on the
surface of each sample (SnF
2
respectively NaBF
4
treated ) and cultured for 3h,
7h and 24h.
Cell nuclei were stained with 5 mMDraq5 diluted 1:1000 in distilled water for 5
min at room temperature.
A B
C D
Visual inspection
demonstrating initial
adherence and proliferation of
fibroblasts.
3h 24 h
SnF2
NaBF4
Fibroblasts
adherence/proliferation
evidence by confocal
microscopy
SnF2
NaBF4
24 h 7 h
SnF2
NaBF4
7 h 24 h 3 h
SEM initial stage of adherence 3h
SnF2
NaBF4
7h
NaBF4
SnF2
24 h
SnF2
NaBF4
MTT assay results showing viable fibroblasts cells
with respect to control and surface treated
alumina/zirconia specimens after 3, 7 and 24 hours of
culture.
The label * indicates p<0.001 versus control, **indicates p<0.01
and *** indicates a p<0.001 with respect to specimen 1.
SnF2
NaBF4
In vivo tests: animal model (rabbit)
50m
Implant
site
Haversian
canal
New bone
proliferation
Interface bone-implant
Haversian
canal
New bone
proliferation
Interface bone-implant
Histology; implant 1 = SnF2 treatment
50m
Implant
site
Haversian
canal
New bone
proliferation
Interface bone-implant
Haversian
canal
New bone
proliferation
Interface bone-implant
50m
Implant
site
50m
50m
Implant
site
Haversian
canal
New bone
proliferation
Interface bone-implant
Interface bone-implant
Haversian
canal
New bone
proliferation
Histology; implant 2 = NaBF4 treatment
Summary
1. We have developed in this work a new strategy for orthopedic/dental
implants based on both concepts improvement: bioactivity and antibacterial
activity by incorporating different concentration of Ag
2
O in PMMA bone
cement followed by collagen electrodeposition.
2. Initial cells attachment is influenced by the silver content in the samples.
3. Collagen layer seems to be an effective agent with respect to fibroblasts
attachment and proliferation.
4. Fluoride-based treatment is proposed to condition the surfaces by improving the
bioactivity of alumina/zirconia composites. SnF2 treatment is more effective than
NaBF4.
5. Both treatments shows similar results, but colonization capability seems to be
promoted by the SnF2 treatment.
6. Morphological details of the fibroblasts attached on the surfaces were
emphasized by SEM showing the formation of a shell-like coating after 24 hours
incubation.
7. Histological images demonstrated the biocompatibility of the treated implants as
no gaps, fibrous tissue, multinucleated cells or inflamation were found at the bone
implant interface. A better bone to implant contact was noticed in the case of SnF2
treatment.
1. Simona Cavalu, V. Simon, C. Ratiu, I. Oswald, S. Vlad, O. Ponta, Alternative Approaches
Using Animal Model for Implant Biomaterials: Advantages and Disadvantages, Key
Engineering Materials Vol. 583 (2014) pp 101-106.
2. Simona Cavalu, V. Simon, F. Banica, I. Akin, G. Goller, Surface modification of
alumina/zirconia bioceramics upon different fluoride-based treatments, Int. J. Appl.
Ceram. Technol.,1-9(2013) DOI:10.1111/ijac.12075.
3. Simona Cavalu, V. Simon, C. Ratiu, I. Oswald,R. Gabor, O. Ponta, I. Akin, G. Goller,
Correlation between structural properties and in vivo biocompatibility of alumina/zirconia
bioceramics, Key Engineering Materials vols. 493-494, 1-6, 2012.
4. Simona Cavalu, V. Simon, I. Akin, G. Goller, Improving the bioactivity and
biocompatibility of acrylic cements by collagen coating, Key Engineering Materials vols.
493-494, 391-3966, 2012.
5. Simona Cavalu, V. Simon, G. Goller, I. Akin, Bioactivity and antimicrobial properties of
PMMA/Ag2O acrylic bone cements collagen coated, Digest J. Nanomaterials and
Biostructures, vol.6/.2 April-June, 779-790, 2011.
6. S. Cavalu, V. Simon, F. Banica, In vitro study of collagen coating by elecrodeposition on
acrylic bone cement with antimicrobial potential, Digest J. Nanomaterials and
Biostructures,vol.6, nr.1 January-March, 87-97, 2010
Acknowledgments:
Romania-Turkey Bilateral Cooperation 2011-2012 and
CNCS-UEFISCDI project PNII-ID-PCE 2011-3-0441 contract nr. 237/2011.
Prof. dr. Viorica Simon Babes-Bolyai
University, Faculty of Physics & Institute of
Interdisciplinary Research in Bio-Nano-
Sciences, Cluj-Napoca, Romania.
Dr. Ioan Oswald and Silviu Vlad,
University of Oradea, Faculty of Medicine
and Pharmaceutics, Oradea, Romania.
Dr. Dumitrita Rugina, USAMV Cluj-
Napoca.
Prof. dr. Gultekin Goller and assist. prof.
Ipek Akin, Istanbul Technical University,
Materials Science Department.