Bioceramics for Load Bearing Applications: Organic/ Inorganic Treatments to Enhance their Bioactivity and Biocompatibility

1. Prof. dr. Simona CavaluFaculty of Medicine and PharmacyUniversity of OradeaROMANIA

2. 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 .

3. Theidealceramicisahighperformancebiocompositethatcombinestheexcellentmaterialpropertiesofaluminaintermsofchemicalstabilityandlowwear,andofzirconiawithitssuperiormechanicalstrengthandfracturetoughness.
Alumina/zirconiaceramics were successfully used in total hip/knee arthroplastyin the last decades.
For dental application: root canal posts, orthodontic brackets, implant abutments and all-ceramic restorations.

4. Bioceramicinteraction with living tissue
Bioinert
Bioactive
Surface modifications and post –synthesis treatments for better performances
Toughandstrongceramicslikezirconia,aluminaoralumina-zirconiacompositesarenotcapableofcreatingabiologicallyadherentinterfacelayerwithboneduetothechemicallyinertnatureofthesetwostableoxides.

5. 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

6. Goal
Inthepresentstudywearefocusedonthepossiblebeneficialeffectoforganiccoating(fibrinogen)andinorganictreatment(fluorinationwithSnF2andNaBF4)withrespecttonewalumina/zirconiabioceramics.
Themainobjectiveistoanalyzethebiocompatibilityofalumina/zirconiaceramicsupontreatmentviainvitroandinvivotests.

7. Materials
Composition: 80%Al2O3–20%YSZ with 5%TiO2addition
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 MPam½ (under a load of 19.6 N)
Vickers hardness 16.7 GPa(under a load of 9.8N).
O. Ormanci, S. Cavalu-Mater SciEng C 40 (2014)

8. FTIR spectroscopy
1200 1000 800 600 400
0
3
5
x
Al
2
O
3
80Al
2
O
3
·20ZrO
2
·xTiO
2
Intensity/ a.u.
Wavenumbers (cm-1)
648
617
465
Modifications of stretching
vibration modes AlO6 octaedra

9. XRD patterns
Al2O3
80Al2O3-20YSZ
80Al2O3-20YSZ +5TiO2
No monoclinic phase ZrO2

10. SEM
80Al2O3-20YSZ
80Al2O3-20YSZ with 5% TiO2
Al2O3

11. Texture of protein (fibrinogen) coating on alumina/zirconiaceramics- electrodeposition

12. Native Fibr
Fibr/specimen 1
Fibr/specimen 2
Native Fibr
Fibr/specimen 1
Fibr/specimen 2
FTIR spectroscopy and deconvolution
αhelix % βsheet% βturns % Random % Side chain% 19.99.2

13. Surface treatment with SnF2and 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

14. Surface treatment- XPS evidence
1200 1000 800 600 400 200 0
F 1s
Al 2s
Zr 3d
Al 2p
C 1s
N 1s
O 1s
Sn 4d Zr 4p F 2s
Sn 3p1
Sn 3d
Zr 3d
N 1s
F 1s
Al 2p
Na 1s
O 1s
C 1s
Intensity (a.u)
Binding Energy (eV)
Sn 3p3
Al 2s
O Auger
Zr 4p
Specimen 2
SnF
2
NaBF
4

15. 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 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 hSnF2NaBF4

16. Fibroblasts adherence/proliferation evidence by confocalmicroscopySnF2NaBF4
24 h
7 hSnF2NaBF4
7 h
24 h
3 h

17. SEM –initial stage of adherence 3hSnF2NaBF4

18. 7hNaBF4SnF2

19. 24 hSnF2NaBF4

20. MTTassayresultsshowingviablefibroblastscellswithrespecttocontrolandsurfacetreatedalumina/zirconiaspecimensafter3,7and24hoursofculture.
The label * indicates p<0.001 versus control, **indicates p<0.01 and *** indicates a p<0.001 with respect to specimen 1. SnF2NaBF4

21. In vivo tests: animal model (rabbit)
Implant 1-SnF2 treatment
Implant 2-NaBF4 treatment
Implant 3-Fibrinogen

22. 50μm
Implant
site
Haversiancanal
New bone proliferation
Interface bone-implant
Haversiancanal
New bone proliferation
Interface bone-implant
50μm
Implant
siteHistology; implant 1 = SnF2 treatmentimplant 2 = NaBF4 treatment
1
2

23. Ca/P= 1.62- 1.80
Haversian canal
Bone morphology after 4 and
8 weeks post -surgery
4 weeks
8 weeks
EDAX

24. XRD spectrum of the femoral bone
0 20 40 60 80 100
0
100
200
300
400
500
600
700
800
900
*
*
AZ A
Z
A
Z
A
A B
Z
A
A
A
Z
A
Z
A
Z
A
A
A
I (a.u.)
2 (deg)
AlZr Biocomposite
Bone/AlZr
Bone
A
T
Z
B

25. Histology: implant 3-bone marrow cells interaction
Implant 3-fibrinogen coating
Goldner’sTrichromestain

26. Histology: implant 3- host bone interaction
Goldner’sTrichromestain
Implant 3-fibrinogen coating

27. SEM/EDX bone-implant interface
Ca/P= 1.62
Ca/P= 1.77
4 weeks
8 weeks

28. Summary
Ceramicspecimenswiththecomposition
80%Al2O3-20%3YSZ+5%TiO2processedbySPSweresurfacetreatedwithSnF2/ NaBF4respectivelyfibrinogenbyelectrodeposition.
Thesurfacemodifications/texturewererevealedbyATR-FTIR,XPSandSEM;itwasdemonstratedthattheSnF2treatmentismoreeffectivethanNaBF4. Proteincharacteristicsarepreservedupondepositionprocedure.
Fibroblastscellscultureinthepresenceoffluorine-treatedspecimensallowedtoassaycelladhesion,cellproliferationandcolonycapabilitybyfluorescenceevaluation.Bothinorganictreatmentsshowssimilarresults,butcellcolonizationcapabilityseemstobepromotedbytheSnF2treatment(cellscultureforfibrinogencoatedisnotshown,workinprogress…..)
MorphologicaldetailsofthefibroblastsattachedonthesurfaceoffluorinetreatedsampleswereemphasizedbySEMshowingtheformationofashell-likecoatingafter24hoursincubation.
Histologicalimagesdemonstratedthebiocompatibilityofthetreatedimplantsasnogaps,fibroustissue,multinucleatedcellsorinflamationwerefoundattheboneimplantinterface.AbetterbonetoimplantcontactwasnoticedinthecaseofSnF2treatment.

29. Animal model-The presence of young, compact lamellar bone and osteocytesnear 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 Haversiancanals) of bone and bone marrow tissue and elemental composition at the interface indicated Ca/P =1.62 -1.77 SummaryConclusions:Organic(proteic)filmorfluorideassurfaceconditioningmightbeanalternativeapproachtoinducethebioactivityandimprovethebiocompatibilityofdensebioceramicsdesignedtoloadbearingbonereplacement(hipjoint,dentalabutments)andtooptimizethebiologicalresponseforspecificapplicationsofbiomedicalimplants.

30. 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 osseointegrationof alumina/zirconiaceramic implants by fluoride surface treatment, Digest Journal of Nanomaterialsand BiostructuresVol. 9, No. 2 (2014) 797 –808.
Simona Cavalu, V. Simon, F. Banica, I. Akin, G. Goller, Surface modification of alumina/zirconiabioceramicsupon 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, ActaPhysicaPolonicaA, 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/zirconiabioceramics, Key Engineering Materials vols. 493-494, 1-6(2012)

31. Acknowledgments: UEFISCDIproject PNII-ID-PCE 2011-3-0441 contract nr. 237/2011 and Bilateral Cooperation RO-TR.
•Prof. dr. VioricaSimonBabes-BolyaiUniversity, Faculty of Physics & Institute of Interdisciplinary Research in Bio-Nano- Sciences, Cluj-Napoca, Romania.
•Dr. Cristian Ratiu, Ioan Oswaldand Silviu Vlad, University of Oradea, Faculty of Medicine and Pharmaceutics, Oradea, Romania.
•Dr. Dumitrita Rugina, USAMV Cluj- Napoca.
•Prof. dr. GultekinGollerand assist. prof. Ipek Akin, Istanbul Technical University, Materials Science Department.