Saša Novak, Nataša Drnovšek, Katja Rade, Spomenka Kobe

Jožef Stefan Institute, Jožef Stefan International Postgraduate School,

Ljubljana, Slovenia

Marko Gradišar

Helipro, Lesce, Slovenia

Outline

Report on the outcome of an FP6 project

Coatings on Ti6Al4V alloy with porous surface layer:

TiO2-anatase coating

Bioactive glass coating

Characterisation

In vivo tests

Project

Multifunctional bioresorbable biocompatible coatings

with biofilm inhibition and optimal implant fixation

IP-SME (2006-2010)

12 partners :

5 research org.

2 laboratories (SME)

4 companies (SME)

1 association

A few pillars

http://fp6.cordis.lu/fp6/home.cfm

Delphi, 2008

Nataša

Katja

Oracle.jpg

Fixation: Cemented Non-cemented

Bone ingrowth

Porous Ti-layer

(

1. Porous Ti-layer:

a) EPD of Ti-powder (KUL-B)

b) VPS – Vacuum plasma spray (Alhenia-CH)

2. TiO2 „bond-coat“:

a) micro-arc anodisation (Uni Bayreuth-D)

b) hydrotermal synthesis (IJS-Slo)

Proposed processes

3. Synthesis of bioactive glass (BAG)

a) melting /milling (KUL-B)

b) polymeric sol-gel (KUL-B; IJS-Slo)

c) particular sol-gel (KUL-B; IJS-Slo)

Depostion of BAG coating

a) EPD (KUL-B; IJS-Slo)

b) vacuum infiltration (IJS-Slo)

Sintering of BAG coating

6. Antibiotic deposition (Hemotec-D)

1. Porous Ti-coating on Ti6Al4V alloy

Not cytotoxic

No HA in simulated body fluid (SBF)

Hydrophobic superhydrofobic

(the cells don‘t enter the pores)

Ti6Al4V Ti(VPS)-layer

VPS (Alhenia-Ch)

EPD (KUL-B)

Ti6Al4V

(Helipro)

Dental

implant

Porous Ti

coated graft

Low-cost technique for coating of

complex shaped implants

Ti-ions

2. TiO2 coating: Hydrothermal treatment Ti-oxide (amorphous) is naturally present on Ti

TNZS

substrate

HT69

HT32 HT50 HT65

HT83

HT80 HT80S

HT84

Dental implant Bone graft with porous Ti layer

TiO2-coating on highly complex-shaped implants

Impedes release of Al and V from Ti6Al4V

(discs 16 mm, h: 1.5 mm) 1 month in MiliQ water (50 ml) + ultrapure NaCl @ 36.5 °C

ICP-MS

D. Cadosch, et al, Biocorrosion and uptake of titanium by human

osteoclasts, J. BIOMEDICAL MAT. RES. A , 2010, 4

Porous Ti-layer on TiAlV HT porous Ti-layer on TiAlV

Improves

wetting

Decoloring of

resazurine dye

UV-induced photocatalitic degradation of organic molecules

HA forms in SBF (Kokubo test) - Ca/P ratio: 1.62

Antibacterial

effect?

Human osteoblast cells: Good viability

S. aureus

1,00E+07

1,00E+08

1,00E+09

1,00E+10

24 48 72

Incubation time (in h)

Cfu

/dis

c

* ** * * *

Staphylococcus epidermidis (1457)

Staphylococcus aureus (ATCC 25923 Bacterial inhibition

µ-T

iO2 (

HT

)

Ti-

all

oy

su

bs

tra

te

n-T

iO2 (

HT

)

Unfinished story…

• Limited reproducibility of the in vitro test (effect of UV light?)

• UV-induced antibacterial effect?

….Starting point for Martina‘s thesis / BioTiNet

Greece … Delphi…

3. Bioactive glass (BAG) coating

Discovered by L. Hench in 1969

BIOACTIVE: due to similarity of the glass components to hydroxyapatite

BIORESORBABLE: the dissolution products nucleate hydroxyapatite

formation at silanol Si-OH groups on the surface.

OSTEOCONDUCTIVE: bond to hard as well as to soft tissue.

BIOINDUCTIVE: ability to repair and to rebuild damaged (hard) tissues

POOR MECHANICAL PROPERTIES !!!

COMPOSITIONS:

SiO2-CaO-Na2O-P2O5 (45S5, melt)

SiO2-CaO-MxOy (M= Zn, Sr, K, Na, P, Mg, ..)

… up to 90SiO2-10 CaO

Comercial BAG

PARTICULATE SOL-GEL

POLYMERIC SOL-GEL

Synthesis methods

MELTING + MILLING

after sintering

After soaking in SBF

for 5 days

Hydroxyapatite formation in simulated body fluid

“SBF test” (sintered pellets)

BACTERIOLOGICAL TEST

after sintering after sintering

24 h

70S30C 53S

24 h

S. aureus 120 h 120 h

7

7,5

8

8,5

9

9,5

10

10,5

11

0 10 20 30 40 50 60

t (h)

pH

70/30 SG-particulate

53S SG-particulate

53S SG-polymeric

45S KUL

(Vivoxid)

fine

powders

coarse

powders

7

7,5

8

8,5

9

9,5

10

10,5

11

0 10 20 30 40 50 60

t (h)

pH

70/30 SG-particulate

53S SG-particulate

fine

powders

sintered

pellets

53S SG-particulate

70/30 SG-particulate

Effect of particles size Effect of sintering

Local pH change during dissolution in water

Impregnation of the porous Ti-layer with BAG

Sintering of the BAG coating

Ti6Al4V

Ti (VPS)

SG-BAG

Ti6Al4V

Ti-porous layer

Nano-Meadow

TEM analysis of the interphase

BAG-coating on Ti6Al4V

substrate

interlayer

glass

1 μm

Ca

Si

O

Ti

Ti

Ti

Si

Al V

60-80 at.% SiO2 10-35 at.% CaO

5-10 at.% TiO2

28-35 at.% Si

72-65 at.% Ti

(Ti5Si3, Ti3Si)

93 at.% Ti

5.4 at.% Al

1.6 at.% V

5

6

Region (close to the substrate)

Crystalline

Larger amount of Ti

What to expect in vivo?

No bacteria! No endotoxins!

Histomorphometric

analysis

10 weeks

rabbits

In-vivo tests

BAG-particular

BAG-polymeric

TiO2-Hydrothermal

TiO2-MAO

Gentamicin

VPS (reference)

14 x

Sample holder

Ti6Al4V graft with

porous Ti layer and

different coatings

Implantation / Explantation

Bone Cylinder

1. Drilling a hole diameter 4.2 mm to achieve pressfit betwen implant and bone

2. Pushing the sterilized cylinder into the hole

3. Closing the wound

~ 10 mm

6 mm

1. Cutting the tibia around the implant 2. Fixation in formaldehid

Implantation (July-August 2011)

Housing for 10 weeks

Explantation (October 2011)

Samples preparation

3) Cutting (vertically)

½: grinding and polishing

- SEM

- EDS (mapping)

- Quantitative analyses

of bone ingrowth - Optical microscopy

- Hystological examination

1) Fixation ½: thinning of 2 slices

2) Mounting (propylene

oxide + EPON / EPON)

HISTOLOGICAL SECTION

SEM

2) Dehidration Staining

(Stevenels blue and

VanGieson picrofuchsin red)

Histological observation

Bone marrow

empty

lacuna NEW

BONE

MATURE

BONE

Ti Ti

Ti

Van Gieson picrofuchsin red

Stevenel’s blue

Porous Ti layers with TiO2 coating

Porous Ti layers without coating (reference)

Bioactive glass

• Bone in the pores

• Good contact with implant

• Mineralised bone in contact

with implant and also in porous

Ti-layer

VPS (reference)

• Mineralised bone in the pores

• Good contact of mineralised

bone with implant

TiO2-HT TiO2/HA-MAO

• No contact between mineralised

bone and implant

• No bone in pores

• A thick layer of unmineralized bone

(osteoid) – something prevents bone

from mineralization – Osteomalacia

(defective mineralization of bone

matrix)

• Osteoclasts !!!

F11

TiO2/HA-MAO

In histological preparations

difficult (if not impossible) to quantify the amount of

bone formed in the porous Ti-layer

Polished cross-sections

SEM, EDS

FEG SEM, EDS (mapping)

Ca, P: indication of bone

Si: remains of BAG

Ti: metal

Al: metal

C, Cl: EPON

Elemental distribution Polished bulk sample

Bone-to-implant contact Presence of bone in the

porous layer

Count: B: points in grey phase (bone)

E: points in Epon (un-field part of the pore)

B (%) = B / (B+E)

VPS (reference)

- 2 samples have cracks between the Ti-coating and

substrate (formed during cutting)

- Moderately high amount of bone in the pores

- Good bone-to-metal contact (surface)

- Half of pores volume filled with bone

- Relatively good contact of bone and implant

- Al at the TiAllV-Ti contact

TiO2 - Hydrothermal treatment

TiO2 anatase coating improves osseointegration of Ti in-vivo

(10 weeks, rabbits)

Ti: No coating TiO2 anatase coating on Ti

Improvement of wetting easier cell entering into the pores

Bone forms also in the deepest parts of the porous Ti-layer

Epon

bone

Ti bone marrow

TiAlV

TiO2-Micro arc anodisation

- No bone in pores

- Almost no contact of bone and implant

- Coating still present in pores (foam-like

appearance)

- No Ca in pores, high P content in the coating

Bioactive glass (particulate sol-gel)

- Relatively high amount of bone in pores

- Relatively good contact of bone and implant

- BAG has dissolved (Si from grinding with SiC paper)

Bone

N. Drnovšek, S. Novak, U. Dragin, M. Čeh, M. Gorenšek, M. Gradišar,

Bioactive glass enhances bone ingrowth into the porous titanium coating on orthopaedic implants,

International Orthopaedics (2012)

47

6

59

45

2

44

0

10

20

30

40

50

60

70

80

90

100

% l

inea

l p

art

of

bon

e-to

-met

al

con

tact

TiO2-anatase coating (HT):

- Simple technique for coating Ti-based materials

- Impedes the release of Al in V release from

the Ti6Al4V alloy

- Improves wetting

- Improves osseointegration in-vivo

- Nano-TiO2 opens the possibility for

UV-induced „self“-sterilisation

(photocatalysis)

BAG-coating (particulate sol-gel):

- Enhances osseointegration

Dental screws

should

er

Standardisation

Clinical tests

In vivo tests

Samples preparation

Development of new/improved biomaterial

?

Nataša Drnovšek

Gregor Murn

Matej Skočaj

Katja Rade

Jozef Vleugels, KUL

(Meddelcoat coordinator)

..and the Meddelcoat team

Marko Gradišar, Helipro d.o.o

Institute for Cell biology, Lj.