Prof. dr. Simona Cavalu

Faculty of Medicine and PharmacyUniversity of Oradea

ROMANIA

Motivation

As the average age of population grows, the need for medical devices to replace damaged or worn tissues increases.

As patients have become more and more demanding regarding esthetic and biocompatibility aspects of their dental restorations .

The ideal ceramic is a high performance biocomposite that combines theexcellent material properties of alumina in terms of chemical stability andlow wear, and of zirconia with its superior mechanical strength and fracturetoughness.

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 restorations.

Bioceramicinteraction with

living tissue

BioinertBioactive

Surface modifications and post –synthesis treatments for better performances

Tough and strong ceramics like zirconia, alumina or alumina-zirconiacomposites are not capable of creating a biologically adherent interfacelayer with bone due to the chemically inert nature of these two stableoxides .

Surface covering

layers/coatings

Biological

response

Cells viability

Cells attachment

Cells proliferation

Surface modification: organic coating/inorganic treatment

Organic: proteins, DNA, sugars.Inorganic: surface blasting , acid etching , fluoride

Goal In the present study we are focused on the possible

beneficial effect of organic coating (fibrinogen) andinorganic treatment (fluorination with SnF2 andNaBF4) with respect to new alumina/zirconiabioceramics.

The main objective is to analyze the biocompatibilityof alumina/zirconia ceramics upon treatment via invitro and in vivo tests.

Materials

Composition: 80%Al2O3 – 20%YSZ with 5%TiO2 addition

Spark plasma sintering method at 1350-1400◦C.

Structural characterization by FTIR and XRD spectroscopy

Morphological details of the surface investigated by SEM

Mechanical properties: Fracture toughness 5.3 MPa m½ (under a load of 19.6 N) Vickers hardness 16.7 GPa (under a load of 9.8N).O. Ormanci, S. Cavalu- Mater Sci Eng C 40 (2014)

FTIR spectroscopy

1200 1000 800 600 400

0

3

5

x

Al2O

3

80Al2O

3·20ZrO

2·xTiO

2

Inte

nsi

ty/ a

.u.

Wavenumbers (cm-1)

64

8

61

7

46

5Modifications of stretching vibration modes AlO6 octaedra

XRD patterns

Al2O3

80Al2O3-20YSZ

80Al2O3-20YSZ +5TiO2

No monoclinic phase ZrO2

SEM

80Al2O3-20YSZ 80Al2O3-20YSZ with 5% TiO2

Al2O3

Texture of protein (fibrinogen) coating on alumina/zirconia ceramics-electrodeposition

Native Fibr

Fibr/specimen 1

Fibr/specimen 2

Native Fibr Fibr /specimen 1 Fibr/specimen 2F

TIR

sp

ect

rosc

op

y a

nd

de

con

volu

tio

n

α helix % β sheet% β turns % Random % Side chain%

19.9 9.2

Surface treatment with SnF2 and NaBF4

- 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- XPS evidence

1200 1000 800 600 400 200 0

F 1

s

Al 2

s

Zr

3d

Al 2

p

C 1

s

N 1

s

O 1

s

Sn 4

d Z

r 4p

F 2

s

Sn 3

p1

Sn 3

d

Zr

3d

N 1

s

F 1

s

Al 2

pNa 1

s

O 1

s

C 1

s

In

tensi

ty (

a.u

)

Binding Energy (eV)

Sn

3p

3

Al 2

s

O A

ug

er

Zr

4p

Specimen 2

SnF2

NaBF4

In vitro test: cells culture Human fibroblast (HLF) seeded in a concentration of 2x104/cm2 cells on the

surface of each sample (SnF2 respectively NaBF4 treated ) and cultured for 3h, 7h and 24h.

Cell nuclei were stained with 5 mM Draq5 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 confocalmicroscopy

SnF2

NaBF4

24 h7 h

SnF2

NaBF4

7 h 24 h3 h

SEM – initial stage of adherence 3h

SnF2

NaBF4

7h

NaBF4SnF2

24 h

SnF2

NaBF4

MTT assay results showing viable fibroblasts cellswith respect to control and surface treatedalumina/zirconia specimens after 3, 7 and 24 hours ofculture.

The label * indicates p<0.001 versus control, **indicates p<0.01 and *** indicates a p<0.001 with respect to specimen 1.

SnF2NaBF4

In vivo tests: animal model (rabbit)Implant 1- SnF2 treatment

Implant 2- NaBF4 treatment

Implant 3- Fibrinogen

50µm

Implantsite

Haversiancanal

New bone proliferation

Interface bone-implant

Haversiancanal

New bone proliferation

Interface bone-implant50µm

Implantsite

Histology; implant 1 = SnF2 treatmentimplant 2 = NaBF4 treatment

1

2

Ca/P= 1.62- 1.80

Haversian canal

Bone morphology after 4 and 8 weeks post -surgery

4 weeks

8 weeks

EDAX

XRD spectrum of the femoral bone

0 20 40 60 80 100

0

100

200

300

400

500

600

700

800

900

*

*

AZAZ

A

ZA

BA

Z

A

AA

ZA

ZA

ZA

AA

I (a

.u.)

2 (deg)

AlZr Biocomposite

Bone/AlZr

Bone

A

T

Z

B

Histology: implant 3- bone marrow cells interaction

Implant 3- fibrinogen coating

Goldner’s Trichrome stain

Histology: implant 3-host bone interaction

Goldner’s Trichrome stain

Implant 3- fibrinogen coating

SEM/EDX bone-implant interface

Ca/P= 1.62

Ca/P= 1.77

4 weeks

8 weeks

Summary Ceramic specimens with the composition

80%Al2O3 - 20%3YSZ + 5% TiO2 processed by SPS were surface treated with SnF2/NaBF4 respectively fibrinogen by electrodeposition.

The surface modifications/texture were revealed by ATR-FTIR, XPS and SEM; itwas demonstrated that the SnF2 treatment is more effective than NaBF4.Protein characteristics are preserved upon deposition procedure.

Fibroblasts cells culture in the presence of fluorine-treated specimens allowedto assay cell adhesion, cell proliferation and colony capability by fluorescenceevaluation. Both inorganic treatments shows similar results, but cellcolonization capability seems to be promoted by the SnF2 treatment (cellsculture for fibrinogen coated is not shown, work in progress…..)

Morphological details of the fibroblasts attached on the surface of fluorinetreated samples were emphasized by SEM showing the formation of a shell-likecoating after 24 hours incubation.

Histological images demonstrated the biocompatibility of the treated implantsas no gaps, fibrous tissue, multinucleated cells or inflamation were found at thebone implant interface. A better bone to implant contact was noticed in thecase of SnF2 treatment.

Animal model- The presence of young, compact lamellar bone and osteocytes near the implant surface indicated good biocompatibility, and certainly the presence of the implant did not disturb the processes of bone formation at the interface, for both organic/inorganic treatment.

Microstructure details (including Haversian canals) of bone and bone marrow tissue and elemental composition at the interface indicated Ca/P =1.62 - 1.77

Summary

Conclusions: Organic (proteic) film or fluoride as surfaceconditioning might be an alternative approach to induce thebioactivity and improve the biocompatibility of dense bioceramicsdesigned to load bearing bone replacement (hip joint, dentalabutments) and to optimize the biological response for specificapplications of biomedical implants.

Related papers:

O. Ormanci, I. Akin, F. Sahin, O. Yucel, V. Simon, Simona Cavalu, G. Goller, Spark Plasma sintered A2O3-YSZ-TiO2 composites: Processing, characterization and in vivo evaluation, Materials Science and Engineering C, 40 (2014) 16-23.

Simona Cavalu, C. Ratiu, O. Ponta, V. Simon, D. Rugina, V. Miclaus, I. Akin, G. Goller, Improving osseointegration of alumina/zirconia ceramic implants by fluoride surface treatment, Digest Journal of Nanomaterials and Biostructures Vol. 9, No. 2 (2014) 797 – 808.

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., 11 [2] 402–411 (2014).

Simona Cavalu, V. Simon, I. Akin, G. Goller, Adherence properties of acrylic bone cement to alumina ceramics designed for clinical application, Acta Physica Polonica A, nr.2,vol.125 (2014) 603-605

S. 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)

Acknowledgments:

UEFISCDI project PNII-ID-PCE 2011-3-0441 contract nr. 237/2011 and Bilateral Cooperation RO-TR.

•Prof. dr. Viorica Simon Babes-BolyaiUniversity, Faculty of Physics & Institute of Interdisciplinary Research in Bio-Nano-Sciences, Cluj-Napoca, Romania.

• Dr. Cristian Ratiu, 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.