Adherence properties of fibroblasts to different bone substitute designed for orthopedic and dental applications

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/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 .

3. The field of tissue engineering is highly interdisciplinary
Bringstogetherpeoplewithknowledgeinmaterialsscience,biochemistry,cellbiology,immunology,andsurgicalexpertisetosolvearangeofopenproblems.
Thesuccessfuldesignoftissue-engineeredconstructsdrivestheneedtodesignnovelbiocompatiblematerialsandstudytheirinteractionswithlivingcells.
Tissueengineeringevolvedfromthefieldofbiomaterialsdevelopmentandreferstothepracticeofcombiningscaffolds,cells,andbiologicallyactivemoleculesintofunctionaltissues.

4. Bioceramicsinvestigated in the present study
Poly(methylmethacrylate)(PMMA) bonecements:
areextensivelyusedincertaintypesoftotalhiportotalkneereplacements
areofpotentialutilitywherevermechanicalattachmentsofmetaltolivingboneisnecessary
Themainfunctionofthecementistoserveasinterfacialphasebetweenthehighmodulusmetallicimplantandthebone,therebyassistingtotransferanddistributeloads.
Alumina/zirconiaceramicsweresuccessfullyusedintotalhip/kneearthroplastyinthelastdecades.
Fordentalapplication:rootcanalposts,orthodonticbrackets,implantabutmentsandall-ceramicrestaurations
isahighperformancebiocompositethatcombinestheexcellentmaterialpropertiesofaluminaintermsofchemicalstabilityandlowwear,andofzirconiawithitssuperiormechanicalstrengthandfracturetoughness.

5. PMMA bone cement Alumina/zirconiabioceramics

6. Motivation
The surface modification and post-synthesis treatment also influences the performances of the bioceramicsdesigned to dental and ortopedicapplications.
According to their interaction with surrounding tissue, bioceramicscan be categorized as ‘‘bioinert’’ or ‘‘bioactive.’’
Tough and strong ceramics like zirconia, alumina or alumina-zirconiacomposites 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 vertebroplastyas for arthroplasty, because is injected directly into the bone instead using as an adhesive agent.

7. Surface modification: organic coating
Theuseofsurfacecoveringlayers(i.e.coatings)provides
methodstocontrolthebiologicalresponsetomaterialsandmaterialdevicesincludingimplantsandprostheses.
Severaltypesoforganicmaterialscanbeusedtogenerateacoatingwithspecificmodulatoryeffectsonthebiologicalresponse.Examplesincludeproteins,DNA, sugars,etc.
Specificbiologicalresponsesthatcanbecontrolledarecellattachmentandbehavior.
Organiccoatingsconsistingofproteinsaregenerallybasedonthepresenceoftheseproteinsattheimplantlocation
[S.Cavalu&all,KeyEngineeringMaterialsVol.583(2014)pp101-106]

8. Surface modification: inorganic molecules
Manydifferenttechniquesarecurrentlyinusetoconditionthesurfacesofabutmentsandfixturesofimplants:surfaceblastingoracidetchingcanincreasetherateandamountofnewboneformationontheimplantsurface.
TheadministrationofcomplexfluoridesascomparedwithNaFsuggeststhepossibilityofusingthemaseffectiveagentsindentalcariespreventioninhumanpopulations.
Forexample,stannousfluorideconvertsthecalciummineralapatiteintofluorapatite,whichmakestoothenamelmoreresistanttobacteriageneratedacidattacks.
[F. Hattab, “The State of Fluorides in Toothpastes,” J. Dent., 17, 47–54 (1989)].

9. Goal
InthepresentstudywearefocusedonthepossiblebeneficialeffectofPMMA/Ag2Ocollagencoatedrespectivelyandsurfacemodificationofalumina/zirconiabioceramicsbyfluoridetreatment
Thesurfacemodificationsofalumina/zirconiabioceramicsareinvestigatedupondifferenttreatmentswithsodiumtetrafluoroborateandstannousfluoriderespectively.
Themainobjectiveistoanalyzethebiocompatibilityofnewbonesubstituteuponsurfacetreatment,viainvitroandinvivotests.

10. 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

11. Biomaterials: PMMA bone cement
Ag2OdopedPMMAisproposedasanalternativetoantibioticloadedcements,silverbeingcapableofkillingover650formsofbacteria,viruses.
Theantimicrobialefficacyofthesecompositesdependsontheirabilitytoreleasethesilverionsfromthesecompositesuponinteractionwithbiologicalfluids.
IthasbeenpreviouslydemonstratedthatbiomimeticcoatingsconsistingofcollagentypeIaresuitablesurfacestoenhancetheirbioactivity,cellattachmentandproliferation[S.Cavalu&all.DigestJournalofNanomaterialsandBiostructures,2010]

12. PMMA/Ag2O bone cement
Asantimicrobialagent,Ag2OparticleswereincorporatedinPMMAwithrespecttothetotalpowderamountinaconcentrationrangingfrom0.1%to4%w/w.
Surface morphology (SEM) of the PMMA/Ag2O specimen surface before any treatment: a) 0.5%Ag2O, b) 1%Ag2O and c) 2%Ag2O.

13. Kinetics of Ag+release from the PMMA specimens with different silver oxide content, during 21 days incubation in Simulated Body Fluid
05101520250.050.100.150.200.250.300.350.400.45 0.10% 0.25% 0.50% 1.00% 2.00% 4.00% Ag+ concentration (mM) Time (days)

14. Possible mechanism of the antimicrobial action of silver ions :
Isnotcompletelyknown
Possibleinteractionwiththyolgroupcompoundsfoundintherespiratoryenzymesofthebacterialcells.
Silverbindstothebacterialcellwallandcellmembraneandinhibitstherespirationprocess.
IncaseofE-coli,silveractsbyinhibitingtheuptakeofphosphateandreleasingphosphate,mannitol, succinate,prolineandglutaminefromtheE-colicells.
Inaddition,itwasshownthatAg+ionspreventDNAreplicationbybindingtothepolynucleotidemolecules,henceresultinginbacterialdeath.

15. 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
640
1140
1240
1436
1722
2950
Absorbance/Arbitrary units
Wavenumber / cm-1
3180
2950
1722
1635
1550
1436
1240
1140
1035
985
640
ATR FTIR spectra recorded on the surfaces of the Ag2O/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).

16. ATR FTIR spectrum of native collagen type I (a), deconvoluted amide I native
collagen (b) and adsorbed collagen to PMMA specimens with 0.5% Ag20 (c),
1% Ag20 (d) and 2% Ag20 (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
1228
Amide III
Wavenumber cm-1
Absorbance (a.u.)
1640
Amide I
1546
Amide II
a)
1600 1620 1640 1660 1680 1700
b)
Absorbance (a.u.)
Wavenumber (cm-1)
1600 1620 1640 1660 1680 1700
Absorbance (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
Absorbance (a.u.)
Collagen
amide I
α helix α helix α helix turns
ν(cm-1) A (%) ν(cm-1) A (%) ν(cm-1) A (%) ν(cm-1) A (%)
native
collagen
1630 28.3 1644 33.2 1665 34.7 1682 3.8
Specimen 1
0.5% Ag2O
1625 40.2 1641 25.5 1657 23.5 1670 10.8
Specimen 2
1% Ag2O
1619 4.2 1637 37.7 1657 43.5 1682 14.6
Specimen 3
2% Ag2O
1630 34.0 1640 44.0 1663 12.0 1673 10.0

17. 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 antiparallelβ-sheet structure (or turns).
As a general behavior, one can observe a shift toward lower frequencies, a decrease in α helix total content and concomitant increase of turn percentage upon adsorption, as a consequence of denaturation.

18. 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 hydroxyapatitecrystals was strongly influenced by the presence of collagen layer, but dependent on the silver oxide concentration as well. [S. Cavalu& all, 2010]

19. Morphology of fibroblasts after 24 h incubation with PMMA specimens. The fibroblasts showed a wide variety of shapes: spread multipolaror round , as well as spindle shaped, elongated cells
0.5%
1%
2%
Human fibroblasts (HSFs) in a density of 2x104cells/cm3were seeded upon each PMMA specimen substrate

20. ResultsshowsviablefibroblastscellswithrespecttocontrolandPMMA/Agwithdifferentconcentrationofsilveroxideafter3,12and24hoursofculture(p<0.05). Initialcellsattachmentisinfluencedbythesilvercontentinthesamples.
Theresultsshowsaprogressivedecreaseinopticaldensityafter3hours,withhighersilverconcentration.Thesamplecontaining1%silveroxideexhibitscomparablebehaviortothatofcontrol(commercialcement).
Fibroblasts viability by MTT assay

21. Biomaterials: Alumina/zirconiaceramic
•Composition : 80%Al2O320%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)

22. 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

23. Surface treatment with fluoride- 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

24. 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.
NaFhas been known to be one of the most effective agents for the treatment of vertebral osteoporosis by its stimulating effect on new bone formation.

25. 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

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

27. SEM –initial stage of adherence 3hSnF2NaBF4

28. 7hNaBF4SnF2

29. 24 hSnF2NaBF4

30. 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

31. In vivo tests: animal model (rabbit)

32. 50μm
Implant
site
Haversiancanal
New bone proliferation
Interface bone-implant
Haversiancanal
New bone proliferation
Interface bone-implantHistology; implant 1 = SnF2 treatment

33. 50μm
Implant
site
Haversiancanal
New bone proliferation
Interface bone-implant
Haversiancanal
New bone proliferation
Interface bone-implant
50μm
Implant
site

34. 50μm
50μm
Implant
site
Haversiancanal
New bone proliferation
Interface bone-implant
Interface bone-implant
Haversiancanal
New bone proliferationHistology; implant 2 = NaBF4 treatment

35. Summary
1.Wehavedevelopedinthisworkanewstrategyfororthopedic/dentalimplantsbasedonbothconceptsimprovement:bioactivityandantibacterialactivitybyincorporatingdifferentconcentrationofAg2OinPMMAbonecementfollowedbycollagenelectrodeposition.
2.Initialcellsattachmentisinfluencedbythesilvercontentinthesamples.
3.Collagenlayerseemstobeaneffectiveagentwithrespecttofibroblastsattachmentandproliferation. 4.Fluoride-basedtreatmentisproposedtoconditionthesurfacesbyimprovingthebioactivityofalumina/zirconiacomposites.SnF2treatmentismoreeffectivethanNaBF4.5.Bothtreatmentsshowssimilarresults,butcolonizationcapabilityseemstobepromotedbytheSnF2treatment. 6.MorphologicaldetailsofthefibroblastsattachedonthesurfaceswereemphasizedbySEMshowingtheformationofashell-likecoatingafter24hoursincubation. 7.Histologicalimagesdemonstratedthebiocompatibilityofthetreatedimplantsasnogaps,fibroustissue,multinucleatedcellsorinflamationwerefoundattheboneimplantinterface.AbetterbonetoimplantcontactwasnoticedinthecaseofSnF2treatment.

36. 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.SimonaCavalu, 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.SimonaCavalu, 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. Nanomaterialsand Biostructures, vol.6/.2 April-June, 779-790, 2011.
6.S. Cavalu, V. Simon, F. Banica, In vitro study of collagen coating by elecrodepositionon acrylic bone cement with antimicrobial potential, Digest J. Nanomaterialsand Biostructures,vol.6, nr.1 January-March, 87-97, 2010

37. Acknowledgments: Romania-Turkey Bilateral Cooperation 2011-2012 and CNCS-UEFISCDIproject PNII-ID-PCE 2011-3-0441 contract nr. 237/2011.
•Prof. dr. VioricaSimonBabes-BolyaiUniversity, Faculty of Physics & Institute of Interdisciplinary Research in Bio-Nano- Sciences, Cluj-Napoca, Romania.
•Dr. 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.