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/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, 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 ofbiomaterials development and refers to the practice ofcombining scaffolds, cells, and biologically activemolecules into functional tissues.

Bioceramics investigated in the present study

Poly (methylmethacrylate) (PMMA)bone cements:

are extensively used in certain typesof 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 materialproperties of alumina in terms ofchemical stability and low wear, andof zirconia with its superiormechanical strength and fracturetoughness.

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 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 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 andmaterial devices including implants and prostheses.

Several types of organic materials can be used to generatea 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]

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, 47–54 (1989)].

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.

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

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]

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 any treatment: a) 0.5%Ag2O, b) 1%Ag2O and c) 2%Ag2O.

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

+ c

on

ce

ntr

atio

n (m

M)

Time (days)

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 DNAreplication by binding to the polynucleotidemolecules, 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

64

0

11

40

12

40

14

36

17

22

29

50

Ab

sorb

an

ce/A

rbitr

ary

un

its

Wavenumber / cm-1

31

80

29

50

17

22

16

35

15

50

14

36

12

40

11

40

10

35

98

5

64

0

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 amideIII 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% 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

12

28

Am

ide

III

Wavenumber cm-1

Abs

orba

nce

(a.

u.)

164

0

Am

ide

I 1

546

Am

ide

II

a)

1600 1620 1640 1660 1680 1700

b)

Abs

orba

nce

(a.

u.)

Wavenumber (cm-1)

1600 1620 1640 1660 1680 1700

Ab

so

rba

nce

(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

Ab

so

rba

nce

(

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 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

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 β-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.

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 2x104 cells/cm3

were seeded upon each PMMA specimen substrate

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).

Fibroblasts viability by MTT

assay

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)

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

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

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 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)

50µm

Implantsite

Haversiancanal

New bone proliferation

Interface bone-implant

Haversiancanal

New bone proliferation

Interface bone-implant

Histology; implant 1 = SnF2 treatment

50µm

Implantsite

Haversiancanal

New bone proliferation

Interface bone-implant

Haversiancanal

New bone proliferation

Interface bone-implant50µm

Implantsite

50µm50µm

Implantsite

Haversiancanal

New bone proliferation

Interface bone-implantInterface bone-implant

Haversiancanal

New bone proliferation

Histology; implant 2 = NaBF4 treatment

Summary

1. We have developed in this work a new strategy for orthopedic/dentalimplants 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-based treatment is proposed to condition the surfaces by improving thebioactivity of alumina/zirconia composites. SnF2 treatment is more effective thanNaBF4.5. Both treatments shows similar results, but colonization capability seems to bepromoted by the SnF2 treatment.6. Morphological details of the fibroblasts attached on the surfaces wereemphasized by SEM showing the formation of a shell-like coating after 24 hoursincubation.7. Histological images demonstrated the biocompatibility of the treated implants asno gaps, fibrous tissue, multinucleated cells or inflamation were found at the boneimplant interface. A better bone to implant contact was noticed in the case of SnF2treatment.

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-BolyaiUniversity, 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.