adherence properties of fibroblasts to different bone substitute designed for orthopedic and dental...

7/21/2019 Adherence properties of fibroblasts to different bone substitute designed for orthopedic and dental applications

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Prof. dr.

Simona Cavalu

Faculty of Medicine and

Pharmacy

University of Oradea

ROMANIA


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Motivation

As the average age of population grows, the need formedical devices/biomaterials to replace damaged or

worn tissues increases.

As patients have become more and more demandingregarding esthetic and biocompatibility aspects oftheir dental/orthopedic restorations .


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The field of tissue engineering is highly

interdisciplinary Brings together people with knowledge in materials

science, biochemistry, cell biology, immunology, andsurgical expertise to solve a range of open problems.

The successful design of tissue-engineered constructsdrives the need to design novel biocompatiblematerials and study their interactions with living cells.

Tissue engineering evolved from the field of biomaterials development and refers to the practice ofcombining scaffolds, cells, and biologically activemolecules into functional tissues.


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Bioceramics

investigated in the

present study

Poly (methylmethacrylate) (PMMA)bone cements:

are extensively used in certain types

of total hip or total kneereplacements

are of potential utility wherevermechanical attachments of metal toliving bone is necessary

The main function of the cement isto serve as interfacial phase betweenthe high modulus metallic implantand the bone, thereby assisting totransfer and distribute loads.

Alumina/zirconia ceramics weresuccessfully used in total hip/kneearthroplasty in the last decades.

For dental application: root canalposts, orthodontic brackets, implantabutments and all- ceramicrestaurations

is a high performance biocompositethat combines the excellent material

properties of alumina in terms ofchemical stability and low wear, andof zirconia with its superiormechanical strength and fracturetoughness.


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PMMA bone cement

Alumina/

zirconia

bioceramics


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Motivation The surface modification and post-synthesis treatment also influences

the performances of the bioceramics designed to dental and ortopedicapplications.

According to their interaction with surrounding tissue, bioceramics

can be categorized asbioinert

or

bioactive.

Tough and strong ceramics like zirconia, alumina or alumina-zirconiacomposites are not capable of creating a biologically adherentinterface layer with bone due to the chemically inert nature of these

two stable oxides .

PMMA cements cannot adhere to existing bone, but thisdisadvantage may not be as pertinent for vertebroplasty as forarthroplasty, because is injected directly into the bone instead using

as an adhesive agent.


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Surface modification: organic coating

The use of surface covering layers (i.e. coatings) providesmethods 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 thebiological response. Examples include proteins, DNA,sugars, etc.

Specific biological responses that can be controlled are cellattachment and behavior.

Organic coatings consisting of proteins are generally basedon the presence of these proteins at the implant location

[S. Cavalu &all, Key Engineering Materials Vol. 583 (2014) pp 101-106]


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Surface modification: inorganic molecules

Many different techniques are currently in use to condition thesurfaces of abutments and fixtures of implants: surface blastingor acid etching can increase the rate and amount of new boneformation on the implant surface.

The administration of complex fluorides as compared with NaFsuggests the possibility of using them as effective agents indental caries prevention in human populations.

For example, stannous fluoride converts the calcium mineralapatite into fluorapatite, which makes tooth enamel moreresistant to bacteria generated acid attacks.[F. Hattab, The State of Fluorides in Toothpastes, J. Dent., 17, 4754 (1989)].


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Goal In the present study we are focused on the possible

beneficial effect of PMMA/Ag2O collagen coatedrespectively and surface modification of alumina /zirconiabioceramics by fluoride treatment

The surface modifications of alumina/zirconiabioceramics are investigated upon different treatmentswith sodium tetrafluoroborate and stannous fluoriderespectively.

The main objective is to analyze the biocompatibility ofnew bone substitute upon surface treatment, via in vitroand in vivo tests.


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Goal

PMMA modified by Ag2Oaddition and collagen coating

80%Al2O3- 20%ZrO2 modified

by surface fluoride treatment

Influence on fibroblastsviability, attachment and

proliferation


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Biomaterials: PMMA bone cement

Ag2O doped PMMA is proposed as an alternative toantibiotic loaded cements, silver being capable of killingover 650 forms of bacteria, viruses .

The antimicrobial efficacy of these composites depends ontheir ability to release the silver ions from these compositesupon interaction with biological fluids.

It has been previously demonstrated that biomimeticcoatings consisting of collagen type I are suitable surfacesto enhance their bioactivity, cell attachment andproliferation [S. Cavalu & all. Digest Journal of Nanomaterials and Biostructures , 2010]


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PMMA/Ag2O bone cement

As antimicrobial agent, Ag2O particles were incorporated inPMMA with respect to the total powder amount in aconcentration ranging from 0.1% to 4 % w/w.

Surface morphology (SEM) of the PMMA/Ag2O specimen surface before anytreatment: a) 0.5%Ag2O, b) 1%Ag2O and c) 2%Ag2O.


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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%

Ag

+

concentration

(mM)

Time (days)


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Possible mechanism of the antimicrobial

action of silver ions : Is not completely known

Possible interaction with thyol group compoundsfound in the respiratory enzymes of the bacterial cells.

Silver binds to the bacterial cell wall and cellmembrane and inhibits the respiration process.

In case of E-coli, silver acts by inhibiting the uptake ofphosphate and releasing phosphate, mannitol,

succinate, proline and glutamine from the E-coli cells. In addition, it was shown that Ag+ ions prevent DNA

replication by binding to the polynucleotidemolecules, hence resulting in bacterial death .


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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/A

rbitraryunits

Wavenumber / cm-1

3180

2950

1722

1635

1550

1436

1240

11401035

985

640

ATR FTIR spectra recorded on the surfaces of the Ag2O/PMMA before andafter collagen electrodeposition. Distinct peaks of collagen: amide I at 1635cm-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).


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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

AmideIII

Wavenumber cm-1

Absorbance

(a.u.)

1640

AmideI

1546

AmideII

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 169-0.000005

0.000000

0.000005

0.000010

0.000015

0.000020 e)

Wavenumber cm-1

Absorbance

(a.u.)

Collagen

amide I helix helix helix turns(cm-1) A (%) (cm-1) A (%) (cm-1) A (%) (cm-1) A (%)

nativecollagen

1630 28.3 1644 33.2 1665 34.7 1682 3.8

Specimen 10.5% Ag2O

1625 40.2 1641 25.5 1657 23.5 1670 10.8

Specimen 21% Ag2O

1619 4.2 1637 37.7 1657 43.5 1682 14.6

Specimen 32% Ag2O 1630 34.0 1640 44.0 1663 12.0 1673 10.0


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Characteristics of FTIR bands

Specific components within the fine structure of amide I adsorbedcollagen is correlated with different states of hydrogen bondingassociated with the local conformations of the alpha chain peptidebackbones.

The highest frequency carbonyl absorption peak represents the

weakest H-bonded system .

The peak located in the higher region, at 1682 cm-1, represent theformation 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 turnpercentage upon adsorption, as a consequence of denaturation.


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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 thepresence of collagen layer, but dependent on the silver oxide concentration as

well. [S. Cavalu& all, 2010]


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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 densityof 2x104 cells/cm3

were seeded uponeach PMMA

specimen substrate


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Results shows viable fibroblasts cells with respect to control and PMMA/Ag withdifferent 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, withhigher silver concentration. The sample containing 1% silver oxide exhibitscomparable behavior to that of control (commercial cement).

Fibroblastsviability by MTT

assay


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Biomaterials:

Alumina/zirconia ceramic

Composition : 80%Al2O3 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)


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Surface treatment with fluoride

ATR FTIR evidence

Fig. 1 ATR FTIR spectra of SnF2 andNaBF4 powders as received from the

supplier .

Fig. 2 ATR FTIR spectra recorded onspecimen surface before and aftertreatment using SnF2 and NaBF4.

Al-O Zr-O


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Surface treatment with fluoride- XPS

evidence

1200 1000 800 600 400 200 0

F1s

Al2s

Zr3d

Al2p

C

1s

N1s

O

1s

S

n4d

Zr4pF2s

Sn3p

1

Sn3d

Zr3d

N

1s

F1s

Al2pN

a1s

O

1s

C

1s

Intensity(a.u)

Binding Energy (eV)

Sn3p

3

Al2s

O

Auger

Zr4p

Specimen 2

SnF2

NaBF4


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Why fluoride?

Administration of complex fluorides suggests thepossibility of using them as effective agents in dentalcaries prevention.

Stannous fluoride converts the calcium mineral apatiteinto fluorapatite, which makes tooth enamel moreresistant to bacteria generated acid attacks.

NaF has been known to be one of the most effective

agents for the treatment of vertebral osteoporosis byits stimulating effect on new bone formation.


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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 5min at room temperature.

A B

C D

Visual inspectiondemonstrating initial

adherence and proliferation offibroblasts.

3h 24 h

SnF2

NaBF4


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Fibroblasts

adherence/proliferation

evidence by confocalmicroscopy

SnF2

NaBF4

24 h7 h

SnF2

NaBF4

7 h 24 h3 h


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SEM initial stage of adherence 3h

SnF2

NaBF4


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7h

NaBF4 SnF2


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24 h

SnF2

NaBF4

MTT lt h i i bl fib bl t ll


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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


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In vivo tests: animal model (rabbit)


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50m

Implantsite

Haversiancanal

New boneproliferation

Interface bone-implant

Haversian

canal



Histology; implant 1 = SnF2 treatment


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50m

Implantsite

Haversiancanal



Haversiancanal


Interface bone-implant50m

Implantsite


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50m50m

Implantsite

Haversiancanal


Interface bone-implantInterface bone-implant

Haversiancanal


Histology; implant 2 = NaBF4 treatment

S


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Summary

1. We have developed in this work a new strategy for orthopedic/dental

implants based on both concepts improvement: bioactivity and antibacterialactivity by incorporating different concentration of Ag2O in PMMA bonecement 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 fibroblastsattachment and proliferation.

4. Fluoride-basedtreatmentisproposed toconditionthesurfacesbyimprovingthebioactivityofalumina/zirconiacomposites. SnF2treatmentismoreeffectivethanNaBF4.5. Bothtreatmentsshowssimilarresults,butcolonizationcapabilityseemstobe

promotedbytheSnF2treatment.6. Morphological details of the fibroblasts attached on the surfaces wereemphasizedbySEMshowingtheformationof ashell-likecoatingafter24hoursincubation.7. Histologicalimagesdemonstratedthebiocompatibilityofthetreatedimplantsasnogaps,fibroustissue,multinucleatedcellsorinflamationwerefoundattheboneimplantinterface.Abetterbonetoimplantcontact wasnoticedinthecaseofSnF2

treatment.


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1. Simona Cavalu, V. Simon, C. Ratiu, I. Oswald, S. Vlad, O. Ponta, Alternative ApproachesUsing Animal Model for Implant Biomaterials: Advantages and Disadvantages, KeyEngineering Materials Vol. 583 (2014) pp 101-106.

2. Simona Cavalu, V. Simon, F. Banica, I. Akin, G. Goller, Surface modification ofalumina/zirconia bioceramics upon different f luoride-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/zirconiabioceramics, Key Engineering Materials vols. 493-494, 1-6, 2012.

4. Simona Cavalu,V. Simon, I. Akin, G. Goller, Improving the bioactivity andbiocompatibility 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 ofPMMA/Ag2O acrylic bone cements collagen coated, Digest J. Nanomaterials andBiostructures, vol.6/.2 April-June, 779-790, 2011.

6. S. Cavalu,V. Simon, F. Banica, In vitro study of collagen coating by elecrodeposition onacrylic bone cement with antimicrobial potential, Digest J. Nanomaterials andBiostructures,vol.6, nr.1 January-March, 87-97, 2010


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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-BolyaiUniversity, Faculty of Physics & Institute ofInterdisciplinary Research in Bio-Nano-

Sciences, Cluj-Napoca, Romania.

Dr. Ioan Oswald and Silviu Vlad,University of Oradea, Faculty of Medicineand Pharmaceutics, Oradea, Romania.

Dr. Dumitrita Rugina, USAMV Cluj-Napoca.

Prof. dr. Gultekin Goller and assist. prof.Ipek Akin, Istanbul Technical University,Materials Science Department.

adherence properties of fibroblasts to different bone substitute designed for orthopedic and dental...

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