Blood Compatibility of Metal Oxide Surfaces Prepared by Metal Plasma Immersion Ion Implantation and Deposition

Maitz, MF; Fitz, T; Pham, MT; Richter, E

Forschungszentrum Rossendorf e.V., Dresden, Germany

BACKGROUND: Metal oxide ceramics, like titania or zirconia are known for their good biocompatibility in or-thopaedic applications. Titanium oxide and iridium oxide already have clinical application as coating on vascular stents and behave superior in clinical studies. This indi-cates also a good blood compatibility of these oxides with minimal clotting activation and minimal induction of smooth muscle cell proliferation as an effect of growth factors from blood platelets. However, an investigation of various metal oxides has not yet been done and also the effect of crystallinity on blood compatibility is not yet clear. These physical and chemical features may be important for blood compatibility because they affect the interaction of proteins and cells with the surface. METHOD: For this study titanium oxide (TiO2), hafnium oxide (HfO2) and iridium oxide (IrO2) was deposited on mirror polished stainless steel (SS) substrates by metal plasma immersion ion implantation and deposition (Me-PIIID) with magnetron sputtering in a reactive oxygen atmosphere. XRD showed that IrO2 can be produced both as nanocrystalline, non textured and as textured film. Both types were used for blood compatibility tests. The film thickness was in a range of 100 nm. SS and mirror pol-ished nitinol (NiTi) were used as a reference. Roughness and contact angle with water were determined of the surfaces. The surfaces were incubated in platelet rich plasma, the number of adherent blood platelets was quantified and the release of platelet derived growth factor (PDGF-AB) and serotonin was measured by ELISA tech-niques as activation markers of blood platelets. Further recalcified human standard plasma was put on the surfaces for a defined time and the production of the F1+2 frag-ment was measured as marker of plasma clotting activa-tion, the terminal attack complex SC5b-9 was measured as marker of complement activation. 125I-Fibrinogen adsorp-tion from standard plasma was measured as surface den-sity. All measurements were done in ten repeats. RESULTS and DISCUSSION: The 10 point roughness of these surfaces was in a similar range of 20 nm for SS, NiTi, HfO2 and the textured IrO2; it was 10 nm for the non-textured IrO2. Major differences were in the wettabil-ity, with lowest contact angle for TiO2 (45°) and HfO2 (59°) and higher angles for SS, NiTi, non textured and textured IrO2 (77°, 71°, 75°, 91°, respectively). This means that all surfaces were very smooth with regards to biological structures. Platelet adherence and platelet activation on the surfaces are shown in Fig. 1. The least platelet adherence was found on the bare metals SS and NiTi, whereas the metal oxides generally show higher, worse values. Activated blood platelets are known to release cytokines with impor-tant messenger functions for other cells. These cytokines, PDGF or serotonin, can be also used to monitor platelet activation. In these tests only minor differences exist for PDGF (Fig. 1) or serotonin release (data not shown).

Surface adsorbed fibrinogen can activate blood platelets and a threshold of 30ng/cm2 for platelet activation has been reported. Here no correlation between fibrinogen adsorption (Fig. 2) and platelet adherence (Fig. 1) was seen, what indicates that additional conformational changes of the molecule are important for the biological effect.

Pla

tele

t Adh

esio

n [%

]

02468

101214

SSNiTiTiOx

HfOx

IrOx non text.

IrOx textured

PD

GF-

AB

[ng/

mL]

0

100

200

300

400

F1+2

Fra

gmen

t [nm

ol/L

]

0.0

0.5

1.0

1.5

2.0

2.5

SS NiTiTiOx

HfOx

IrOx n

on text.

IrOx te

xtured

Fibr

inog

en [n

g/cm

2 ]

0

20

40

60

80

100

120

Fig. 1: Platelet adherence and activa-tion on different metal and metal oxide surfaces. The box plots indicate median, quartils and range of 10 measurements.

Fig. 2: Fibrinogen adsorption on the surfaces and rate of clotting cascade activation (F1+2 fragment forma-tion)

The non textured IrO2 which allows better entanglement of proteins also absorbs the highest amount of fibrinogen and induces significantly the highest activation of up-stream parts of the clotting cascade (measured as release of F1+2 peptide), and of the complement cascade (data not shown). However the correlation between fibrinogen adsorption and cascade activation does not generally exist. None of the biological parameters correlated with any of the physical surface properties like roughness, wettability or surface energy. CONCLUSION: The crystalline and textured IrO2 shows significantly less interaction with proteins and protein cascades, therefore surfaces with higher crystallinity seem to have a better blood compatibility. Otherwise correla-tions between physical surface properties and biological effects are limited. This puts more focus on chemical and biochemical functional groups of a surface, what is cur-rently under investigation. The untreated metal surfaces with natural oxide film often show a better blood compatibility than the thicker metal oxide films, but they are less corrosion resistant and there-fore may behave inferior in the clinical studies.

Blood Compatibility of Metal Oxide Surfaces Prepared by

Metal Plasma Immersion Ion Implantation and Deposition

Manfred F. Maitz, Temenoujka Fitz, Minh-Tan Pham, Edgar Richter

Forschungszentrum RossendorfDresden, Germany

Objectives• Production of oxides of the transition metals

– Titanium– Hafnium– Iridium

as coatings on stainless steel coupons and stents• Physical characterization of the coatings• Hemocompatibility testing

– Plasmatic systems– Blood platelets

Preparation of the Coatings• Sputter Cleaning in Argon or Oxygen Plasma

(2kV, 2000 Hz, 10min)• Deposition by magnetron sputtering in a

reactive oxygen atmosphere (0.5 Pa, 20min)• PIII (20kV, 2000-3000Hz) only initially or during

whole deposition time.

IrOx Coating on Stainless Steeloutside inside

un-e

xpan

ded

expa

nded

Adherent coating also after expansion can be achieved on real vascular stents

Surface Morphology

SS NiTi TiOx

HfOx IrOx non textured IrOx textured

Surface Roughness

SS NiTiTiOx

HfOx

IrOx non text.

IrOx textured

Rou

ghne

ss S

z[nm

]

0

5

10

15

20

25

Sz Roughness measured between 5 highest and 5 deepest points on 5x5µm2 AFM scans

Red blood cell, blood platelet and lymphocyte

FibrinogenLow roughness, more in the range of biomolecules than cellular structures

X-Ray Diffraction

20 30 40 50 60 70

HfO2 Hf

Inte

nsity

(a. u

.)

2 Θ20 30 40 50 60 70

TiO2

Inte

nsity

(a. u

.)

2 Θ

30 40 50 60 70

IrO2 no texture3 nm

a)

IrO2 Ir

Inte

nsity

(a. u

.)

2 Θ 30 40 50 60 70 80 90

b) IrO2(101) texture andamorphous phase2.3 nm

IrO2 Ir

Inte

nsity

(a. u

.)

2 Θ

TiOx HfOx

IrOx non textured IrOx textured

X-Ray Diffraction

{100}none28% {002}rest {100}

Orientation

2.3±0.5 nm3.0±0.5 nm6 ± 2 nm2-3nmGrain size

47% IrO235% austenite18% ferrite

38% IrO238% austenite24% ferrite

35% HfO245% Hf10% austenite10% ferrite

Rutile TiO2 (anatase) austeniteferrite

Composition

IrOx text.

IrOx non text.

HfOxTiOx

Surface Energy

Inte

rfac

e En

ergy

[mN

/m]

010203040506070

total

SSNiTiTiOx

HfOx

IrOx non text.

IrOx textured

01020304050

dispersivepolar

Contact Angle(Water)

SS NiTiTiOx

HfOx

IrOx non text.

IrOx textured

Con

tact

Ang

le

0°

20°

40°

60°

80°

100°

Plasma Proteins – Clotting Cascade

SSNiTi

TiOxHfOx

IrOx non te

xt.

PIII IrO

x textured

Fibr

inog

en [n

g/cm

2 ]

0

20

40

60

80

100

120

Fibrinogen Adsorption from whole plasma

Prothrombin F1+2 Fragment

F1+2

Fra

gmen

t [nm

ol/L

]

0.0

0.5

1.0

1.5

2.0

2.5

SSNiTi

TiOxHfOx

IrOx non text.

IrOx texturedF1

+2 M

ean

Ran

k

0

2

4

6

Non-textured IrOx adsorbs highamount of fibrinogen and strongly activates the clotting cascade

Complement Cascade

SC5b

-9 C

ompl

ex [µ

g/L]

200

400

600

800

1000

1200

1400

SSNiTi

TiOxHfOx

IrOx non text.

IrOx texturedSC

5b-9

Mea

n R

ank

0

2

4

6

Terminal Attack Complex SC5b-9 of the Complement Cascade

• High activation on non-textured IrOx

• Low activation on othermetal oxides

Blood Platelet Adhesion

Blood platelet adherence on a surface as haemostatic principle

• Generally lower adherence on bare metal with natural oxide layer

% P

late

let A

dher

ence

0

2

4

6

8

10

12

14

SSNiTi

TiOxHfOx

IrOx non text.

IrOx textured

Mea

n R

ank

0

2

4

6

Platelet Activation – PDGF-Release

PDG

F-A

B [n

g/m

L]

0

100

200

300

400

SSNiTi

TiOxHfOx

IrOx non text.

IrOx texturedPD

GF

Mea

n R

ank

0

2

4

6

α-granule

PDGF-AB release from α-granules of blood platelets• Minor differences• Good ranks for Nitinol and TiOx

Platelet Activation – Serotonin Release

Sero

toni

n [µ

g/m

L]

0

10

20

30

40

50

60

SSNiTi

TiOxHfOx

IrOx non text.

IrOx textured

Sero

toni

n M

ean

Ran

k

0

2

4

6

dense granule

Serotonin release from dense granules of blood platelets• Minor differences• Similar trends as PDGF-AB

Rank OverviewAveraged Rank ofTwo platelet parameters

Platelet AdherencePDGF Release

Two plasma parametersF1+2 FragmentSC5b-9

• Best overall hemocompatibility for TiOx and Nitinol with naturally grown TiO2

• Significantly better hemocompatibility of textured than non-textured IrOx

SSNiTi

TiOxHfOx

IrOx non text.

IrOx textured

Mea

n To

tal R

ank

0

1

2

3

4

5

6

Correlation of Physical Parameters with Blood Compatibility

• No significant correlation between – any bio parameter– the rank scoreand any physical parameter.

→ Higher Impact of Chemistry?• Most prominent difference between the smooth

non-textured and the textured IrOx– Improved entanglement and activation at the non-

textured, multi-oriented IrOx is discussed– Further influences?

Comparison of non-textured and textured IrOx

Non-textured IrOx Textured IrOx

Ir : O = 1 : 3 Ir : O = 2 : 3

AES Element Profiles

Sputter time [s]0 200 400 600 800

Con

cent

ratio

n [a

t-%]

0

20

40

60

80

100COCrFeNiIr

Sputter time [s]0 100 200 300 400 500 600

Con

cent

ratio

n [a

t-%]

0

20

40

60

80COCrFeNiIr

Comparison of non-textured and textured IrOxXPS Spectra

Binding Energy [eV]

260270280290300310320330

C 1

s - I

r 4d

[arb

. U]

IrIrClxIrClx CCH

Binding Energy [eV]

161820222426283032343638

O 2

s [a

rb. U

]

IrO2

Binding Energy [eV]

50556065707580

Ir 4f

[arb

. U]

IrO2IrClx

Binding Energy [eV]

515520525530535540545550555

O 1

s [a

rb. U

]

IrOx non texturedIrOx textured

IrO2H2O

hydrocarbons

Conclusions• Inside and outside adherent TiO2 and IrO2

coatings can be produced on vascular stents • TiO2: Slight improve of blood compatibility

compared to metal oxides and bare stainless steel

• Higher activation of plasmatic cascades on non textured IrOx than on textured– Unclear whether direct consequences of crystallinity

or indirect due to increased adsorption of other material.

AchnowledgementFritz Prokert

Helfried ReutherJan Weber

André ReichelElke Quaritsch

Heike Zimmermann

Supported by: Boston Scientific SCIMED