Adhesion and Activation of Platelets And

8/3/2019 Adhesion and Activation of Platelets And

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A d h e s i o n a n d a c t i v a t i o n o f p l a t e l e t s a n dp o l y m o r p h o n u c l e a r g r a n u l o c y t e c e lls a t T i O 2 sur faces

H,~KAN NYG REN, CEC ILIA ERIKSSON, an d JUKKA LAUSMAA

GOTEBO RG, SWEDEN

The in it ia l react ions o f two T iO2 sur faces w i th b loo d w ere invest iga ted by sh or t - t ime

e xp o s u r e to ca p i l l a r y b l o o d a n d a n a l ys is o f su r fa ce - a d s o r b e d p la sm a p r o te in s a n d

su r fa ce - a d h e r i n g ce l l s b y u s in g immu n o f l u o r e sce n ce te ch n iq u e s . An t i b o d ie s d i -

r e c te d a g a in s t p l a te l e t me m b r a n e a n t i g e n a n d P- se le c ti n w e r e u se d to v isu a l ize

p la te le t a d h e s io n a n d a c t i va t i o n . Ac r i d i n e o r a n g e a n d a n t i- C D 1 Ib w e r e u se d to

d e te c t a d h e s io n a n d a c t i va t i o n o f p o l ym o r p h o n u c le a r g r a n u lo cy te s ( PMN s). An t i -

b o d ie s a g a in s t t h r o mb o sp o n d in w e r e u se d a s ma r ke r s fo r p l a te l e t s - g r a n u le s . Th e

f l u o re s c e n c e i nt e ns i ty w a s q u a n ti t a te d b y c o m p u t e r - a i d e d i m a g e a n al ys is . C o m -me r c ia l l y p u r e , p o l i sh e d sh e e t t it a n iu m w a s o x id i ze d i n tw o d i ff e r en t w a ys : ( I ) t h e

n a tu r a l o x id e w a s d i sso l ve d w i th h yd r o f l u o r i c a c id a n d a n e w o x id e l a ye r w a s

grow n by o x ida t ion in n it r ic ac id , o r (2) anne a l ing was p er fo rm ed a t 700 ° C in a ir .

A u g e r e l e c t r o n s p e c t r o s c o p y a n d x - r a y p h o t o e l e c t r o n s p e c t r o s c o p y s h o w e d t h a t

b o th su r fa ce s h a d s im i l a r co m p o s i t i o n co n s i s ti n g o f T iO2 co v e r e d b y a ca r b o n a -

ce o u s su r fa ce co n ta m in a t i o n l a ye r. The th i ckn e ss o f t h e o x id e l a ye r w a s 4 n m o n th e

a c id - o x id i ze d su r fa ce a n d 3 9 n m o n th e a n n e a le d su r fa ce . Op t i ca l p r o f i lo me t r y a n d

s c a n n i n g e l e c t r o n m i c r o s c o p y s h o w e d t h a t th e a c i d - o x i d i z e d s u r fa c e w a s r o u g h

a n d th e a n n e a le d su r fa ce w a s smo o th . Th e f i b r i n o g e n /p r o th r o mb in - th r o mb in r a t io

in the in i t ia l p ro te in f i lm d i f fe red be tween the sur faces. The number o f adher ingp la te le ts w a s l a r g e r a t t h e su r fa ce w i th a h ig h su r fa ce co n ce n t r a t i o n o f a d so r b e d

f ib r inogen. P la te le t act iv a t ion (CD62) and p r iming o f PMN s (CD 1 b ) were a lso

s ign i f i can t ly h igher on the ac id -ox id ized sur face . The resu l ts ind ica te tha t non-se l f

r e co g n i t i o n o f b i o ma te r i a l s i s a n a r r a y o f t ra n s ie n t r e a c t io n s c o mp r i s i n g p r o te in -mate r ia l , p ro te in -ce l l , and ce l l - ce l l in te ract ions. ( J Lab C l in M ed 1997; 129 :35-46)

Abbreviations: AES = Au ger electron sp ectrosco py; D-PBS = Dulbec co's phosp hate-bufferedsaline solution containing physiolog ic concentrations of K, Ca, a nd Mg; FITC = fiuoresceinisothiocyan ate; PMN = polym orphonu clear granulocy te; XPS = x-ray photoem ission spec lros-co p y

From the Departm ent of Anatomy and C ell Biology, University

of G6teborg; and the D epartment of Applied Physics, Chalm ersUniversity of Technology.

Supported by grants from the Sw edish Medical Research C ouncil(06235), The Research Council of Engineering Sciences, and T heSwedish Biom aterial Consortium.

Subm itted for publication April 23, 1996; revision submitted July10, 1996; acce pted J ul y 30, 1996.

Reprint requests: Hfikan Nygren, PhD , Department of A natomyand Cel l Biology, Med icinaregatan 5, S-413 90 G 6tebo rg, Sweden.

Copyright © 1997 by M osby-Year Book, Inc.

0022-2143/97 $5.00 + 0 5/1/76968

Al m o s t a n y m e d i c a l d e v i c e i n t r o d u c e d i n to

t h e h u m a n b o d y w i l l i n t e r a c t i n i ti a l ly w i t hb l o o d , a c o n t a c t t h a t w i t h o u t e x c e p t i o n s

l e a d s t o s u c h r e a c t i v e c h a n g e s i n t h e t i s s u e s a s b l o o d

c o a g u l a ti o n , i n f l a m m a t i o n , a n d f o r m a t i o n o f c o ll a g-

e n o u s t i s s ue . T h e i n f l a m m a t o r y r e a c t i o n a s s u c h ,

b e i n g p a r t o f t h e h e a l i n g p r o c e s s a s w e l l a s o f r e j e c -

t i o n , c a n t h u s n o t b e u s e d t o f o r e c a s t t h e p e r f o r -

m a n c e o f a m a t e r i a l i n c o n t a c t w i t h l i vi n g t i ss u e . A n

u n d e r s t a n d i n g o f t h e d i f f e r e n c e b e t w e e n n o n - s e l f

r e c o g n i t i o n l e a d i n g t o h e a l i n g a n d t h a t l e a d i n g t o

r e j e c t io n c a n b e a c h i e v e d o n l y w h e n t h e p a t h w a y s o f

3 5



J Lab Cl in Med3 6 Nygren, Eriksson, an d Lausmaa Jan uar y 1997

t h e s e p r o c e s s e s a r e d e f i n e d . I t is a ls o o f c o n s i d e r -

a b l e i n t e r e s t i n b i o m a t e r i a l s r e s e a r c h t o g a i n a n

i n c r e a s e d u n d e r s t a n d i n g o f h o w t h e s e p r o c e s s e s a r e

i n f l u e n c e d b y d i f f e re n t b i o m a t e r i a l s u r f a c e p r o p e r -

t i es ( c h e m i c a l c o m p o s i t i o n , m i c r o s t r u c t u r e a n d m o r -

p h o l o g y , w e t t a b i l i t y , e t c . ) .

B i o m a t e r i a l s i n c o n t a c t w i t h b l o o d i n s t a n t l y a c -

q u i r e a s u r f a c e l a y e r o f h o s t p l a s m a p r o t e i n s 1 a n d

m o s t li k e ly o t h e r m a c r o m o l e c u l e s . T h e s u r f a c e

p r o p e r t i e s o f t h e m a t e r i a l d e c i d e t h e c o m p o s i t i o n

a n d s t r u c t u r e o f t h e p r o t e i n f i l m , w h i c h t h e n s e ts o u t

t h e c o n d i t i o n s f o r c e l l a d h e s i o n a n d a c t i v a t i o n . 2 P r e -

v i o u s i n v e s t i g a t o r s h a v e r e p o r t e d t h a t a l b u m i n c a n

p a s s i v a t e a s u r f a c e , 1 t h a t c o m p l e m e n t a c t i v a t io n c a n

r e s u l t i n t h e n e u t r o p h i l r e c r u i t m e n t t o s u r f a c e s , 3 '4

a n d t h a t f i b r i n ( o g e n ) p l a y s a r o l e i n t h e a c u t e i n -

f l a m m a t o r y r e s p o n s e . 5 A s t r o n g c o r r e l a t i o n h a s

b e e n f o u n d i n se v e r a l s t u d i e s b e t w e e n t h e a d s o r p -

t i o n o f fi b r i n o g e n a n d s u r f a c e a d h e s i o n o f p l a t e -

l e ts . 6 -s A d h e s i o n o f p l a t e l e t s d o e s n o t n e c e s s a r i l y

c o m p r i s e a c t i v a t i o n o f t h e c e ll s, a n d a c t i v a t i o n in

i t s e l f i s n o t a w e l l - d e f i n e d t e r m . P l a t e l e t a d h e s i o n t o

s u r f a c e - i m m o b i l i z e d f i b r i n o g e n r e s u l t s i n a c t i v a t io n

o f t h y r o s i n e p h o s p h o r y l a s e k i n a s e a n d r e o r g a n i z a -

t i o n o f t h e c y t o s k e l e t o n b u t n o t i n s e c r e t i o n o f s e -

r o t o n i n , 9 g e n e r a l l y c o n s i d e r e d a m a r k e r o f p l a t e l e t

a c t i v a t i o n .

T h e a i m o f th e p r e s e n t s t u d y w a s to e l u c i d a t e t h e

m e c h a n i s m s o f a c t i v a t i o n o f s u r f a c e - a d h e r i n g p l a t e -l et s a n d P M N s . A d h e s i o n w a s m e a s u r e d b y a g e n -

e r a l m a r k e r f o r t h e c e ll , a n d a c t i v a t i o n w a s m e a -

s u r e d b y sp e c i fi c m a r k e r s - - t h a t i s , a n t i - C D 6 2 P

a n t i b o d i e s f o r d e t e c t i o n o f P - s e le c t i n o n p l a t e l e t

s u r f a c es 1°-12 a n d a n t i - C D l l b a n t i b o d i e s f o r d e t e c -

t i o n o f a n u p - r e g u l a t e d c o m p l e m e n t r e c e p t o r a t t h e

s u r f a c e o f P M N s . 13-15

T h e w i d e s p r e a d a n d s u c c e ss f u l cl in i ca l u s e o f t i-

t a n i u m a s a n i m p l a n t m a t e r i a l m o t i v a t e s a st u d y o f

t h e i n i t i a l r e a c t i o n s o f t h is m a t e r i a l w i t h b l o o d t o

i n v e s t i g a te w h i c h h u m o r a l a n d c e l l u l a r d e f e n s e s y s -

t e m s m a y b e a c t i v a t e d o n a s u r f a c e o f a m a t e r i a l t h a ti s k n o w n t o b e w e l l i n t e g r a t e d i n t o t i ss u e s . 1 6'1 7 I n

t h is w o r k w e h a v e i n v e s t i g a t e d t w o d i f f e r e n t m o d i -

f i ca t i o n s o f t i t a n i u m s u r f ac e s , a n d w e h a v e f o u n d

t h a t t h e y e l i c i t q u i t e d i f f e r e n t b i o l o g i c r e s p o n s e s i n

c o n t a c t i n g h u m a n b l o o d .

M E T H O D S

S u r f a c e p r e p a r a t i o n . C o m m e r c ia l l y p u r e ( 9 9 .6 % )

s h e e ts o f t i t a n i u m w e r e o b t a i n e d f r o m a l o c a l m a n u f a c -

tur e r . P ieces 8 × 8 × 1 m m 2 w er e w ash ed thr ee t imes in

d i c h l o r o m e t h a n e f o l l o w e d b y a c e t o n e a n d w a t e r t o r e -

move gr oss or ganic contamina t ion . A f i r s t g r oup w as an-nea led a t 700 ° to 800 ° C to a p ur p le in te r f e r ence co lor . A

second gr oup w as immer sed in 10% hydr of luor ic ac id ( 5

minutes ) to r emo ve the na t ive ox ide fi lm . The p la tes w er e

r insed thr ee t imes in 100 vol o f w a te r and im me r sed in

concent r a ted n i t r i c ac id f or 10 minutes to f or m a new

oxide layer ( samples r e f e r r ed to as acid-oxidized). T h e

p r e p a r e d s a m p l e s w e r e i m m e d i a t e ly i m m e r s e d i n b o i l ed ,dis t i l led water and s tored in plas t ic vials before use.

Surface characterization. T h e s u r fa c e t o p o g r a p h y a n d

r oughness of tw o samples of each type w er e qua l i t at ive ly

and quant i t a t ive ly eva lua ted by scanning e lec t r on mic r os -

copy and by opt ica l p r of i lomet r y ( Topscan 3D ) , r espec-

t ive ly . The scanning e lec t r on mic r oscop y w as ope r a ted in

t h e s e c o n d a r y e l e c tr o n m o d e , a n d m i c r o g ra p h s w e r e t a k e n

f r om sever a l r ando mly se lec ted a r eas on each sample . The

pr of i lom et r y da ta w er e acqui r ed f r om 245 × 245 ~m ar eas

a t a r eso lu t ion of 1 txm . The f o l low ing topogr aphica l

p a r a m e t e r s s w e r e m e a s u r e d : s u r f a c e r o u g h n e ss ( g r m s ) ,

me an la te r a l d i s tance be tw ee n sur f ace asper i t ie s ( SJ , a n d

sur f ace en la r gement ( r a t io be tw een ac tua l sur f ace a r ea

and p r o jec ted a r ea , Sdr) .

The samples w er e ana lyzed w i th r espec t to sur f ace com-

pos i t ion and oxide th ickness by x- r ay photo emiss ion spec-

t r o s co p y a n d A E S .

The A ES ana lyses w er e do ne in a scanning A uger mi -

cro pro be (P HI 660; Physical Electronics Division, Perkin

Elm er Cor p . , Eden P r a i rie , Minn . ) w i th a pr imar y bea m

cur r en t o f 250 nA and ener gy of 3 .0 keV . Sur vey spec t r a

f r o m f i v e p o i n t s o f 20 0 ~ m d i a m e t e r w e r e r e c o r d e d f r o m

o n e s a m p l e o f e a c h p r e p a r a t i o n w i t h a n a n a l y ze r re s o l u -

t ion of 0 .6%. H igh- r eso lu t ion sp ec t r a ( ana lyzer r eso lu t ion

0 . 3 % ) o f t h e T i a n d O p e a k s w e r e a l so a c q u i r e d f r o m o n ep o i n t o n e a c h s a m p l e . D e p t h p r o fi l es w e r e m e a s u r e d a t

o n e p o i n t o f e a c h s a m p l e b y u s i n g 2 k e V A r i o n s f o r

sput te r ing , w i th a ca l ib r a ted sput te r ing r a te of 6 .0 nm/m in

( f or T iO 2) . The base pr es sur e in the scanning A uger

m i c r o p r o b e e q u i p m e n t w a s 2 × 1 0 9 m b a r d u r in g t h e

A ES sur f ace ana lyses and 1 × 10 8 m bar dur ing depth

pr of i l ing . The r e la t ive a tomic concent r a t ions w er e ca lcu-

la ted f r om the r e la t ive peak he ights a f te r cor r ec t ion by

tab ula ted sensitivity fa ctors . 19

The X PS ana lyses w er e ca r r ied o u t in com mer c ia l , cus -

tom er - mod i f ied X PS equipm ent ( 5000-C; Phys ica l E lec -

t r on ics) . Sur vey spec t r a w er e acqu i r ed f r om an 800 ~ m

d i a m e t e r a r e a o n t w o s a m p l e s o f e a c h t y p e b y u s i n gm o n o c h r o m a t e d A 1 K a r a d i a t i o n a n d a p a s s e n e r g y o f

187 .85 eV . Q u ant i f ica t ion of the sur vey spec t r a w as don e

b y m e a s u r in g t h e i n t e g ra t e d p e a k a r e a s a f t e r b a c k g ro u n d

sub tract ion a nd by us ing the atom ic sensi tivity facto rs

pr ovided w i th th e sof tw ar e o f the ins t r ument . 2° H igh-

r eso lu t ion spec t r a of the T i 2p , O l s , and C l s r eg ions

w er e r ec or ded by us ing mon och r om ated A 1 K c~ r ad ia t ion

and a pas s ener gy of 5 .85 eV to ob ta in in f or mat ion ab out

the chemica l s ta te of these e lements .

E x p o su re o f c a p i l la r y b l o o d a n d i m m u n o f l u o r e s c e n c e .

A ppr oximate ly 20 ml of cap i l l a r y b lood f r om hea l thy hu-

man donor s w as p laced in dr ops on the sur f aces and

contac ted f or t ime per iods r anging be tw een 5 seconds and32 minutes in a hum if ied cham ber a t 37 ° C . The b lood w as



J Lab C l i n Me dVolum e 129, Num ber 1 Nygren , Er iksson,and Lausm aa 37

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Fig. 1. The effect of e thanol f ixat ic , on the amoun t of surface-bound CD62 and CD11b (perce nt

coverage). CD 62 was measured af ter 8 minutes of blood exposure and the am ount of C D llb was

measu red a f ter 32 minutes of bloo d exposure of two t i tanium surfaces wi th d i f ferent surface p roper t ies .

t h e n w a s h e d o f f D - P B S , a n d t h e s a m p l e w a s p l a c e d o n a

c o o l i n g p l a t e a t 0 ° C . T h e s p e c i m e n s w e r e i n cu b a ~ :e d f o r 2 0

m i n u t e s w i t h s p e c i f ic r a b b i t a n t i - h u m a n a n t i b o d i e s ( D a -

k o p a t t s , C o p e n h a g e n , D e n m a r k ) d i r e c t e d a g a i n s t p l a t e l e t

m e m b r a n e a n t ig e n s ( p a n - a n t ib o d y ) o r c o m p l e m e n t f a c t o r

C l q , f i b r in o g e n , a n d p r o t h r o m b i n / t h r o m b i n , f o l lo w e d b y

i n c u b a t i o n f o r 2 0 m i n u t e s w i t h F I T C - c o n j u g a t e d a n t i -

r a b b i t i m m u n o g l o b u l i n a n t i b o d i e s . C e l l a c ti v a t io n w a s d e -

t e c t e d b y in c u b a t i o n w i th m o u s e m o n o c l o n a l a n t i b o d i e sa g a in s t h u m a n C D l l b o r C D 6 2 ( S e ro t e c, O x fo r d , E n -

g l a n d ) , a n d p l a t e l e t v e si c l es w e r e d e t e c t e d b y a n t i b o d i e s

a g a i n s t t h r o m b o s p o n d i n ( B o e h r i n g e r , M a n n h e h n ) . A f t e r

b e i n g r i n s e d i n D - P B S , t h e s p e c i m e n s w e r e i n c u b a t e d f o r

2 0 m i n u t e s w i t h F I T C - c o n j u g a t e d a n t i - m o u s e i m m u n o g l o b -

u l i n a n t ib o d i e s . C o n t r o l e x p e r i m e n t s w e r e p e r f o r m e d b y

e x c l u d in g t h e f i r s t a n t i b o d y i n c u b a t i o n . T h e s p e c i m e n s w e r e

r i n s e d aga i n a n d m o u n t e d i n D a b c o ( F l u k a B i o c h em i k a ,

S w i t ze r l and) an d f ina l ly cov e red w i th a g l a s s cove r s l i p .

S t a i n i n g w i t h a c r i d i n e o r a n g e w a s p e r f o r m e d b y i m m e r -

s i o n o f th e s p e c i m e n f o r 3 m i n u t e s i n t o a 0 . 0 1 % s o l u t i o n o f

a c r i d i n e o r a n g e ( S i g m a C h e m i c a l C o . , S t L o u is , M o . ) i n 0 .0 6

m o l / L p h o s p h a t e b u f f e r , p H 6 .0 . A l l s p e c i m e n s w e r e k e p t

f r o z en a t b e l o w - 2 0 ° C u n t il e x a m i n a t io n a n d p h o t o g r a p h -

ing in a f l uo rescence m ic roscope (3R S ; Z e i s s ) . T he f i lm

s p e e d , m a g n i f i c a ti o n , a n d e x p o s u r e t i m e w e r e k e p t c o n s t a n t.

T h e p h o t o g r a p h s w e r e s c a n n e d a n d d i g i ti z e d i n a P o -

l a r o i d s c a n n e r , a n d t h e b r i g h t n e s s o f t h e p i c t u r e s w a s

a n a ly z e d w i th t h e A d o b e P h o t o s h o p p a c k a g e o n a P o w e r

M a c i n t o s h 8 1 0 0 / 8 0 d e s k t o p c o m p u t e r . B a c k g r o u n d c o n -

t r o ls w e r e u s e d a s th e z e r o l e v el . T h e m e a n b r i g h t n e s s o f

t h e p i c t u r e , o r t h e s u r f a c e c o v e r a g e o f f l u o r e s c e n c e w i t h

a n i n t e n s i t y a b o v e b a c k g r o u n d , w a s u s e d a s t h e q u a n t i t a -t iv e m e a s u r e o f t h e a m o u n t o f b o u n d a n t i b o d i e s.

E t ha no l f i x a t ion . I n o n e e x p e r i m e n t , p i e c e s o f s i l v e r -

c o l o r e d t i t a n i u m w e r e f i x e d i n a b s o l u t e e t h a n o l f o r 1 0

m i n u t e s a t - 2 0 ° C a f t e r b l o o d e x p o s u r e a n d w e r e r e h y -

d r a t e d b e f o r e b e i n g p u t o n t h e c h i l l e r p l a t e . T h e p u r p o s e

w a s t o s e e w h e t h e r t h e r e w a s a d i f f e r e n c e in t h e a m o u n t

o f C D 6 2 a n d C D 1 1 b b e t w e e n s u r f a c es f ix e d i n a b s o l u t e

e t h a n o l a n d s u r f ac e s p u t d i r e c t ly o n t o t h e c o o l i n g p la t e .

T h e r e s u l ts a r e s h o w n i n F i g . 1 , w h e r e t h e a m o u n t o f

C D 6 2 a f t e r 8 m i n u t e s o f b l o o d e x p o s u r e a n d t h a t o f

C D 1 1 b a f t e r 32 m i n u t e s o f b l o o d e x p o s u r e w e r e m e a s n r e d

a s p e r c e n t c o v e r a g e o f t h e s u r f a c e . T h e s u r f a c e f i x e d in

e t h a n o l s h o w e d a s i g n i f ic a n t l y ( p < 0 . 0 1 ) h i g h e r C D 6 2

c o v e r a g e , 1 . 1 % _+ 0 . 2 % , t h a n t h e s u r f a c e n o t f i x e d , 0 .6 %



J Lab C l i n M ed

38 Ny gren , E ri ks son , and Lau s m aa J an ua ry 1997

Fig. 2. Scanning electron micrographs of titanium surfaces. Bar = 0.1 mm. A, Piece of titanium washed

in organic solvents and h eate d at 700 ° C in air. B, Piece o f titanium was hed in organic solvents, etched in

hydrofluoric acid, and oxidized in concentrated nitric acid.

Table I. Opt ical surface profi lometry results

Preparation R,ms (l~m)* Sz (l~m)t Scx (l~m)~ Sdr§

HNO3-o x idized 1.90 26.9 14.0 1.79

Annealed 0.45 7.40 10.7 1.17

*RMS surface roughness o ver the measured area (245 x 245 i~m, lateral

resolution 1 ~m).

tAvera ge value of the heights of the 5 highest peaks and the depths of the

5 dee pest valleys, with respect to a center plane, within the measured area.

:l:Average spacing between profile peaks in the mean plane along the x-

direction (chosen perpendicular to polishing groo ves on annealed surface,

direction irrelevant for HNO3 sample).

§Surface enlargement factor-- that is, ratio between develo ped surface

area and projected area.

_+ 0.1%. A significant difference (p < 0.02 ) in C D ll b

coverage was also observed between the surfaces: 8.8% -+

3.0% for the ethanol-fixed samples and 11.7% + 2.5% for

the no nfixed samples. Beca use the differe nces in coverage

were small between the two surfaces and the results con-

tradictory, w e decide d to proceed w ithou t ethano l fixation

in the other experiments.

Statistical evaluation. The differences between means

were compared by using Student's t test. All experimentswere ru n in triplicate, bu t norma lized values of CD62 an d

CDllb were repeated to give n = 12.

RESULTS

Surface topography. Fig . 2 shows scanning e lect ron

microscopy images of the two sur faces . The an-

neale d sur face (Fig . 2, A) has a re la t ively smo oth

topog raphy , wi th g rooves in the mic ron r ange tha t

are preferent ia l ly or ien ted a long one d irect ion . The

acid-oxid ized sur face (Fig . 2 , B) has a qui te d i f feren t

sur face topography, wi th a h igh degree o f roughne ss

on the 1 to 10 p.m scale . The overal l appe aranc e o fth is sur face ind icates an iso tropic e tch ing , resu l t ing

in a facet ted sur face expos ing predominant ly low-

index c rys tal f aces . The quan t i t a tive m eas u rem en t s

by op t ica l p ro f i lome t ry o f the s u r face topog raphy o f

the three sur faces , summarized in Table I , ver i f ied

the impress ion f rom scanning e lect ron microscopy:

the a nnea led sur face was s ignif icantly smo othe r and

cons equen t ly had a lower s u r face a r ea than the

aci d-ox idiz ed surfa ce (Rrms, 0.45 Ixm vs 1.90 txm,

respect ively ; sur face en largement fac tor , 1 .17 vs

1.79, respectively).Surface chemical composition. The re la t ive sur face

concen t r a t ions o f the de tec ted e l emen t s , a s de te r -

mined by AES and X PS s u rvey ana lys es, a r e s um-

mar ized in Tables I f and I I I . Both sur faces were

foun d to cons is t main ly of a TiO2 sur face oxide

covered by a ca rbon -domina ted s u r face con tamina -

t ion l aye r wi th mino r amoun t s o f o the r impur i t ie s

(Si, C1, A1, N, F, Na, etc.) . The s imilar results ob-

t a ined fo r the d i f f e r en t a reas ana lyzed by AES in-

d ica te tha t the two p repa ra t ions l ead to s amp les

with uniform sur face compo s i t ion . The carbon levels

de tec ted by AES a r e in the r ange o f 20% to 25% ,with very s imilar mean values for the ac id-oxid ized

s amp les and the annea led ones (24% and 26% ,

respect ively) . For the XPS analyses the detected

carbon levels were in the range 35% to 45%, with a

s omewha t lower mean va lue fo r the ac id -ox id ized

s amp les (38% ) a s compared wi th the annea led s am-

ples (44%). The d if ference in absolu te levels be-

tween A ES and XPS i s a t l ea st pa r t ly due to d i f f e r-

ences in re la t ive sens i t iv i ty fac tors a nd sur face

sens i t iv i t ies of the two techniques , and i t does not

necessar i ly ref lec t rea l d i f ferences in absolu te con-

cen t r a t ions . The am oun t o f n i t r ogen de tec ted byXPS on the annealed sample is about twice as h igh



J L a b C l i n M e d

V o l u m e 1 2 9, N u m b e r 1 N y g r e n , E ri ks s on , a n d L a u s m a a 3 9

T a b l e I I. R e l a t i v e c o n c e n t r a t i o n s ( a t o m i c % ) o f e l e m e n t s d e t e c t e d i n A E S a n a l y s e s , i n t e n s i t y r a t i o s b e t w e e n

3 9 0 e V a n d 4 2 0 e V p e a k s , a n d i n t e n s i t y r a t i o s b e t w e e n Ti ( 4 2 0 e V ) a n d O p e a k s

l(390ev)/l(420ev) I0/I~Preparation Area T i O C P Si Cl AI F rat io rat io

H N O 3 - o x i d i z e d 1 2 0 , 7 5 3 . 8 2 4 - - 0 . 6 0 . 3 0 . 5 - - 0 . 5 7 2 . 6 12 2 0 . 4 5 3 .1 2 4 . 9 - - 0 , 5 - - 0 . 5 0 , 6 0 . 5 6 2 . 6 1

3 1 9 , 6 5 3 2 6 - - - - 0 , 2 - - 1 . 2 0 . 5 5 2 . 7 0

4 2 0 . 3 5 2 .1 2 6 , 7 - - 0 . 6 0 , 3 - - - - 0 , 5 3 2 . 5 6

5 2 1 . 4 5 1 .1 ' 2 7. 4 - - - - 0 . 2 - - - - 0 . 5 7 2 . 3 9

A n n e a l e d 1 2 1 . 0 5 5 , 8 2 1 . 2 0 . 5 0 . 7 - - 0 . 7 - - 0 . 5 9 2 . 6 6

2 2 0 . 8 5 4 . 3 2 4 . 3 0 . 3 - - - - 0 . 3 - - 0 . 5 9 2 . 6 1

3 2 1 . 0 5 0 .1 2 6 . 9 0 . 6 0 . 4 - - 1 . 0 - - 0 . 5 8 2 . 3 9

4 2 1 . 0 5 3 . 8 2 4 , 0 0 . 6 - - - - 0 , 5 - - 0 , 5 8 2 . 5 6

5 2 0 .1 5 2 . 3 2 5 , 7 0 . 2 0 . 6 - - 1 . 0 - - 0 , 5 7 2 , 6 1

T a b l e I II . R e l a t i v e c o n c e n t r a t i o n s o f e l e m e n t s d e t e c t e d i n X P S a n a l y s e s

Preparation Sam ple no. f i O C N Si F Sn

H N O 3 - o x i d i z e d 1 1 4 , 4 4 4 . 1 3 5 .1 3 . 7 0 . 8 0 . 6 1 . 3

H N O 3 - o x i d i z e d 2 1 0 . 8 4 0 . 3 4 1 . 5 5 . 4 0 . 7 0 . 8 0 . 5

A n n e a l e d 1 1 0 .1 3 7 . 3 4 3 . 4 8 . 9 0 . 3 - - - -

A n n e a l e d 2 9 .1 3 7 .2 4 4 .4 8 .1 1 .1 - - - -

as tha t on the ac id -ox id ized sur face , which i s in

q u a l i t a t i v e ag reemen t w i t h t h e o b s e rv ed d i f f e r en ces

i n th e r a ti o b e t w e e n t h e 3 9 0 e V ( b e c a u s e o f o v e r -

l app ing Ti and N s ignal s) and th e 420 eV (on ly Ti )p eak s i n t h e A E S an a l y s es . T h e o x i d e t h i ck n es s e s

m e a s u r e d b y A E S d e p t h p r o f il in g w e r e 3 n m f o r th e

ac i d -o x i d i zed s u r f ace an d 3 9 n m fo r t h e an n ea l ed

surface .

T h e X PS h i g h - r e s o l u t i o n s p ec t r a (F i g . 3 ) r ev ea l

f u r t h e r a n d m o r e d e t a i l e d i n f o r m a t i o n a b o u t t h e

ch emi ca l s t a t e o f t h e d e t ec t ed e l em en t s . T h e s h ap e ,

p o s i t i o n ( - 4 5 9 eV ) , an d s p i n -o rb it s p li tt in g (5 .8 eV )

o f t h e T i 2 p s p ec t r a (F ig . 3 , A ) a r e i n c l o s e ag ree -

m en t w i t h t h o s e o f s t o i ch i o me t r i c T iO 2 2° -2 4 fo r b o t h

s amp l es . T h e d i f f e r en ces i n p eak w i d t h an d en e rg y

b e t w e e n t h e t w o s u r f a ce s a r e a c o n s e q u e n c e o f t h ed i f f e r en ces i n ro u g h n es s an d o x i d e t h ick n es s fo r t h e

t w o s amp l es , w h i ch g i v e ri s e t o a l o w er r e s o l u t i o n fo r

the ac id -ox id ized sample and a smal l charg ing- in -

d u ced e n e rg y s h if t f o r t h e an n e a l ed s amp l e . N o s i g n s

of o ther ox idat ion s t a tes such as carb ide , n i t r ides , o r

s u b o x i d es a r e d e t ec t ed , ex cep t f o r a mi n o r me t a l l i c

co n t r i b u t i o n a ro u n d 4 5 4 eV fo r t h e ac i d -o x i d i zed

s amp l e . T h i s m e t a l li c co n t r i b u t i o n i s cau s ed b y t h e

b u l k me t a l b e l o w t h e s u r f ace o x i d e an d a r i s e s b e -

cau s e t h e s u r f ace o x i d e i s t h i n n e r t h a n t h e i n fo rma-

t ion dep th o f the analys i s.

Th e O l s spe ct ra (Fig . 3, B) are q ual i t a t ive lys i mi l a r f o r b o t h s u r f aces an d a r e d o mi n a t ed b y a

p eak a ro u n d 5 3 0 . 5 eV t h a t can b e a s s i g n ed t o o x y -

g en in TiO2 20-24 Ad di t ional spect ra l fea tu res are

o b s e rv ed a ro u n d 5 3 2 . 5 eV an d 5 3 3 . 5 eV , an d t h ey

d i f f e r s o mew h a t i n ab s o l u t e an d r e l a ti v e i n t en s it i e sb e t w een t h e t w o s amp l es . T h e l a t t e r f ea t u r e s a r e

a t t r i b u t ed t o o x y g en b o u n d t o ca rb o n an d h y d ro g en

in th e a d s o rb e d o ve r la y e r ( e.g ., - O H , - C O O H ,

- C = O ) .

The C l s spect ra (Fig . 3 , C) are a l so qual i t a t ive ly

s imi lar , wi th a main peak around 285 .5 eV. Th i s

p eak i s a s s i g n ed t o h y d ro ca rb o n s p r e s en t i n t h e

ad s o rb ed o v e r l ay e r. I n ad d i ti o n , t w o r e l a ti v e ly b ro ad

fea t u r e s , c en t e r ed a ro u n d 2 8 7 eV an d 2 8 9 eV , a r e

d ea r l y d e t ec t ed . T h e r e l a ti v e i n t en s it i e s o f th e s e

peaks are s ign i f i can t ly d i f feren t fo r the two samples .

Fu r t h e r s t u d ie s a r e n eces s a ry t o s a f e l y a s s ig n t h e s ep eak s , b u t t h ey a r e o b v i o u s l y cau s ed b y v a r i o u s o r -

g an i c fu n c t i o n a li t ie s i n t h e ad s o rb ed o v e r l ay e r (mo s t

l i k e l y ca rb o n b o u n d ma i n l y t o O b u t a l s o t o n i t r o -

g en ) . N o s i g n s o f ca rb i d e w ere d e t ec t ed .

Final ly , the N l s peak (Fig . 3 , D) cons i s t s o f one

s in g l e co m p o n en t ( b u t w i th d i f f e r en t ab s o l u t e i n t en -

s i t i e s ) f o r b o t h s amp l es . T h e b i n d i n g en e rg y ( -4 0 1

eV) sugges t s tha t the n i t rogen ex i s t s main ly in an

o rg an i c fo rm.

P r o te i n a d s o r p t i o n a n d p l a t e l e t a d h e s i o n . A f t er 5 sec-

o n d s o f b l o o d ex p o s u re , t h e am o u n t o f su r f ace -

a d s o r b e d p r o t h ro m b i n , C l q , a n d f i b ri n o g e n w a sme as u red a s me an b r i g h t n es s o f t h e p i c tu r e s , w h e re



J Lab Clin Med40 N yg r e n ,Eriksson, and Lausm aa Janu ary 1997

t ~, ' ~_

t -

.d

C

oO

4 . . J

c :. m

o

' ' ' I . . . . I

( a )

, . , , " " " ; e t :

i i ,

4 7 0

29 2

I . . . .

Ti 2p

I r , , , I ,

465 460 455 450

I I 1 I

( b ) . / : ', .. 0 ls

i

536

, I , , I , , I ; " , , - ( ~ F ~ ' ~ T ~ , ,~v~¢7I ~ ~ ~ -' -'"~"-'~'"""-'

534 532 530 528 526

I i I

290 288 286

Bind ing energy(eV)

I

C l s

284 282

( d )I I I I

N l s

/ " " i ' "

,

/ " !

406 404 402 400 398 396

Binding energy (eV)

Fig . 3 . H igh- re so lu t ion XPS spec t ra o f (A) T i 2p, (B) O l s , (C) C ls , and (D) Nls r eg ions fo r annea le d

titanium ( s o l i d l i n e ) and HNOa-oxidized titanium ( d o t t e d l i n e ) . The T i2p spectra are in close agreementwith those of stoichiometric Y i O 2 2 0 " 2 4 or both samples. The O ls spectra of the two surfaces show that adominating peak around 530.5 eV can be assigned to oxy gen. The additional peaks around 532.5 eV and533.5 eV are attr ibuted to oxygen bound to carbon.

2 5 6 is t h e l e v e l r e a c h e d w h e n a ll pi x e ls r e c o r d m a x -

i m u m b r i g h t n e s s ( F ig . 4 , A ) . S t a i n i n g w i t h a n t i p r o -

t h r o m b i n a n t ib o d i e s s h o w e d a m o r e t h a n 3 t i m e s

h i g h e r l e v e l o f b r i g h t n e s s o n t h e a n n e a l e d t i t a n i u m

( 9 7 .0 + 0 . 2 ) t h a n o n t h e a c i d - o x i d i z e d s u r f a c e (2 4 .5 _+

2 .9 ), w h e r e 2 5 6 i s t h e m a x i m u m b r i g h t n e s s l e v e l.

S t a in i n g w i t h a n t i - C l q a n t i b o d i e s a l s o r e s u l te d i n

h i g h e r l e v e l s o f fl u o r e s c e n c e b r i g h t n e s s o n t h e a n -

n e a l e d s u r f a c e ( 6 7 _+ 4 . 6 ) t h a n o n t h e a c i d - o x i d i z e d

sur f ac e ( 46 .3 _+ 2 .6) .

A d i f f e r e n t d i s t ri b u t i o n w a s s e e n w i t h a n t i b o d -i e s a g a i n s t f i b r i n o g e n , w h e r e t h e a c i d - o x i d i z e d

t i t a n i u m s h o w e d m o r e f l u o r e s c e n c e i n t e n s i t y

( 6 4 .1 _+ 0 . 7 ) t h a n t h e a n n e a l e d s u r f a c e ( 4 7 . 0 _ +

0 .2 ). T h e a m o u n t o f a d h e r i n g p l a t e l e t s w a s m e a -

s u r e d a f t e r 5 s e c o n d s o f b l o o d e x p o s u r e a s p e r c e n t

c o v e r a g e o f t h e s u r f a c e ( F i g . 4 , B ) . T h e r e s u l t s

s h o w e d t h a t t h e a m o u n t o f p l a t e l e t s a d h e r i n g t o

t h e a c i d -o x i d i z e d t it a n i u m ( 6 . 5 % + 0 . 3 % ) w a s

t w i ce a s h i g h a s t h e a m o u n t a d h e r i n g t o a n n e a l e d

t i t a n i u m ( 3 . 0 % -+ 0 . 3 % ) .

Act ivat ion of p l a t e l e t s . T h e a m o u n t o f C D 6 2 a n d

t h r o m b o s p o n d i n w a s m e a s u r e d a s p e r c e n t c o v e r a g ea f t e r b l o o d e x p o s u r e f o r 2 , 4 , a n d 8 m i n u t e s ( F ig . 5 ).



A 1 0 0

8 0

d

+1

u )

( 9

J ~O ~

, i

In

mp .

60

40

T

2 0

0

P r o t h r o m b i n

T

C l q

[ ] A

[ ] B

F i b r i n o g e n

J L a b C l i n M e d

V o l u m e 1 2 9, N u m b e r 1 N y g r e n , E rik ss on , a n d L a u s m a a 4 1

B

c iuJ

4-1

>

0

7

6

5

4

3

2

0

[ ] A

[ ] B

T

P l a t e l e t s

F i g . 4 . A , T h e a m o u n t o f s u r f a c e - a d s o r b e d p r o t h r o m b i n / t h r o m b i n , C l q , a n d f i b r i n o g e n m e a s u r e d b y

i m m u n o f l u o r e s c e n c e a n d c o m p u t e r - a i d e d i m a g e a na ly si s ( A d o b e P h o t o s h o p a n d N I H i m a g e ) . T h e l e v e l

256 represen ts m a xim u m br igh tness . Typ ica l backgrou nd leve ls a re 3 to 4 and have been sub t rac ted f rom

the p rese n ted da ta . M ean an d SD are g iven (n = 3 ) . A , Annea led ; B , HNO 3-ox id ized . B , Surface coverage

o f p l a t e l e ts m e a s u r e d b y i m m u n o f l u o r e s c e n c e w i th a p o l y c lo n a l p a n - p la t e l e t a n t i b o d y a f t e r 5 s e c o n d s o f

exposure o f t i tan ium to b lood . A , Ann ea led ; B , HNO3-ox id ized .



42 Nygren, Eriksson, an d Lausm aa'J Lab C l i n Med

Janua ry 1997

-t-I

O }L_

>0

o

2

1 . 6

1 . 2

0 . 8

0 . 4

0

[ ] A C D 6 2

[ ] B C D 6 2

[ ] A t h r o m b o s p o n d i n

[ ] B t h r o m b o s p o n d i n

O iiiT

2 4 8T i m e ( m i n )

Fig. 5. Th e am ount of C D62 and thrombospondin on the titanium surfaces measured as percent surfacecoverage of f luorescence above background level, as a function of time of blood-material exposure. A,Annealed; B, HNO3-oxidized.

T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e ( p > 0 . 0 5) i n

c o v e r a g e o f t h r o m b o s p o n d i n b e t w e e n t h e t w o s u r-

f a c e s o r w i t h t i m e o n e i t h e r o f t h e s u r fa c e s .

T h e a m o u n t o f C D 6 2 d i f f e re d w i th t i m e a s w e l l a s

b e t w e e n t h e t w o s u r f a c e s . T h e r e w a s n o s i g n i f i c a n t

d i f f e r e n c e (p > 0 .0 5 ) in c o v e r a g e o f C D 6 2 o n a n -

n e a l e d t i t a n i u m b e t w e e n 2 a n d 4 m i n u t e s , a n d t h ec o v e r a g e w a s v e r y l ow . B e t w e e n 4 a n d 8 m i n u t e s t h e

a m o u n t o f a d s o r b e d C D 6 2 h a d i n c r e a se d m o r e t h a n

1 6 t i m e s . F o r a c i d - o x i d i z e d t i ta n i u m , t h e c o v e r a g e o f

C D 6 2 w a s v e r y l o w d u r i n g t h e f ir s t 4 m i n u t e s . A t 8

m i n u t e s t h e c o v e r a g e o f C D 6 2 w a s m o r e t h a n 6 0

t i m e s h i g h e r t h a n t h e c o v e r a g e a f t e r 2 m i n u t e s.

W h e n t h e t w o s ur f ac e s w e r e c o m p a r e d t h e r e w a s

n o s i g n i f i c a n t d i f f e r e n c e ( p > 0 . 0 5 ) i n c o v e r a g e o f

C D 6 2 b e t w e e n h e a t - t r e a t e d a n d a c i d - o x i d i z e d t i t a -

n i u m a f t e r 2 m i n u t e s . A f t e r 4 m i n u t e s t h e a m o u n t o f

a d s o r b e d C D 6 2 w a s s i g n i f ic a n t l y l o w e r (p < 0 . 0 1 ) o n

t h e a c i d - o x id i z e d s u r f ac e . A f t e r 8 m i n u t e s o f e x p o -s u r e t o b l o o d t h e r e w a s a s i g n i f i c a n t l y ( p < 0 . 0 1 )

h i g h e r c o v e r a g e o f C D 6 2 o n a c i d - o x i d i z e d ti t a n i u m

t h a n o n a n n e a l e d t i t a n i u m .

A dhe s i on a nd a c t i v a t i on o f ne u t roph i l s . A f t e r 8

t h r o u g h 3 2 m i n u t e s o f b l o o d - m a t e r i a l c o n t a c t , th e

a m o u n t o f C D 1 1 b a n d a c r id i n e o r a n g e - s t a i n e d l e u -

k o c y te s w e r e m e a s u r e d a s p e r c e n t c o v e r a g e o f t h e

s u r f a c e ( F i g . 6 ). A f t e r 8 m i n u t e s , o n l y a fe w l e u k o -c y t es w e r e d e t e c t e d o n b o t h s u r fa c e s . T h e m o r p h o l -

o g y o f th e s t a i n e d n u c l e i s h o w e d c l e a rl y t h a t t h e

a d h e r e n t c e ll s w e r e P M N s . A f t e r 1 6 m i n u t e s o f

e x p o s u r e , t h e c o v e r a g e o f l e u k o c y t e s h a d i n c r e a s e d

e q u a l l y o n t h e t w o s u r f a ce s to 2 . 2 % _ 0 . 3 % . A t 3 2

m i n u t e s o f e x p o s u r e t o b l o o d , l e u k o c y t e s o n t h e

a n n e a l e d t i t a n i u m h a d i n c r e a s e d t o 3 . 2 % _ 0 . 7 % .

T h e c o v e r a g e o f l e u k o c y t e s o n a c i d - o x id i z e d t i ta -

n i u m i n c r ea s e d m o r e t h a n 3 ti m e s b e t w e e n 1 6 a n d

3 2 m i n u t e s , u p t o 7 . 2 % +_ 0 . 9 % .

T h e c o v e r a g e o f C D 1 1 b i n c r ea s e d c o n t i n u o u sl y

w i t h t i m e . O n a n n e a l e d t i t a n i u m , n o C D 1 1 b w a sd e t e c t e d a f t e r 8 m i n u t e s , a n d t h e c o v e r a g e w a s l o w



J Lab Clin MedVolume 129, Number 1 Nygren, Eriksson, and Lausmaa 43

+1

o }

t n

>O

( 3

1 5

1 0

5

0

[ ] A leucoc ytes

[] B l e u c o c y t e s

[] A CD1 l b

[ ] B CD11b

8 1 6 3 2T i m e ( m i n )

Fig. 6. The amount of CD1 1b and acridine orange fluorescence on the titanium surfaces measured aspercent surface coverage of f luorescence above background level, as a function of time o f blood-materialexposure. A, An nealed; B, HNO3 -oxid~ed.

a f t e r 1 6 m i n u t e s . A f t e r 3 2 m i n u t e s , t h e c o v e r a g e o f

C D l l b h a d i n c r e a s e d t o o n l y 0 . 5 % +_ 0 . 1 % .

O n a c id - o xi d iz e d ti t an i u m , C D l l b w a s p r es e n t

a f t e r 8 m i n u t e s a n d s h o w e d a c o v e r a g e o f 0 . 2 % _+

0 . 1% . A f t e r 1 6 m i n u t e s, t h e c o v e r a g e o f C D l l b w a s

6 0 t i m e s h i g h e r o n a c i d - o x i d i z e d t i t a n i u m t h a n o n

a n n e a l e d t i t a n i u m , a n d a f t e r 3 2 m i n u t e s , c o v e r a g e

w a s m o r e t h a n 2 5 t i m e s h i g h e r .N o r m a l i z e d v a l u e s o f C D 6 2 a n d C D 11 b . W e wanted to

m a k e s u r e t h a t t h e d i f f e re n c e s b e t w e e n t i le t w o

s u rf a c es re g a r d i n g C D 6 2 a n d C D l l b w e r e re a l an d

n o t a r e s u l t o f m o r e c e l ls p r e s e n t o n o n e s u r f a c e

t h a n o n t h e o t h e r. T h u s c o v e r a g e o f C D 6 2 w a s

n o r m a l i z e d t o t h e a m o u n t o f p l a t e l e t s a d h e r i n g t o

t h e s u r f a c e s a f t e r b l o o d e x p o s u r e f o r 8 m i n u t e s .

C o v e r a g e o f C D l l b w a s n o r m a l i z e d to th e n u m b e r

o f le u k o c y t e s a f t e r 3 2 m i n u t e s o f b l o o d e x p o s u r e .

T h e r e s u l t s a r e s h o w n i n T a b l e I V ( n = 1 2 ). A l -

t h o u g h t h e c o v e r a g e o f C D 6 2 w a s s i g n i f i c a n t l y

h i g h e r ( p < 0 . 0 1 ) o n t h e a c i d - o x i d i z e d t it a n i u m t h a n

o n t h e a n n e a l e d t i t a n i u m a f t e r 8 m i n u t e s , t h e d i f-

T a b l e IV. Normalized coverage of t itanium

surfaces with CD6 2/pa n pl atel et after 8 minutes of

blood exposure, and CD11b/acridine orange

after 32 minutes of b lo od expos ure (me an _+ SD),

n=12

A nnea l ed A c i d -ox i d i z ed

CD62 0.4 _+ 0.1 0.6 _+ 0.1Platelets 5.3 -+ 0.8 3.6 + 0.5

CD62/Platelets 0.08 0.2CD11b 0.5 _+ 0,1 11.7 _+ 2.5Acridine 3.2 + 0.7 7.2 + 0.9

CD11 b/acridine 0.2 1.6

t e r e n c e w a s s m a ll . H o w e v e r , b e c a u s e m o r e p l a t e l e t s

a d h e r e d t o t h e a n n e a l e d t i t a n i u m t h a n t o th e a c i d-

o x i d i z ed s u r f a c e a f t e r 8 m i n u t e s o f b l o o d e x p o s u r e ,

p l a t e l e t s o n t h e a c i d - o x i d i z e d t i t a n i u m s e c r e t e d 2 . 5

t i m e s m o r e C D 6 2 p e r c e l l .

T h e a m o u n t o f C D 1 1 b w a s 23 t im e s h i g h e r o n t h e



J Lab C l in Me d44 Nygren, Er iksson, an d Lausm aa Jan uary 1997

acid-oxidized surface after 32 minutes exposure

time, but also more than twice as many leukocytes

were present. So after normalization, there was still

a difference between the two surfaces, with the acid-

oxidized surface having the higher percent coverage

of CD ll b per cell, but the difference was 8 times

after normalization.

DISCUSSION

The results of the present study show that a com-

plex biofilm is formed within a few seconds of blood-

material contact. The protein content of the film

differs between materials and is sensitive to small

variations in the physical properties of the material.

The surface characterization of the modified tita-

nium surfaces show that the oxide stoichiometry

(TiO2) and the composition of the adsorbed surface

contamination layer are quite similar for two types

of surfaces, with the exception of some minor quan-

titative differences in the exact composition of the

outer contamination layer. The two surfaces do,

however, di ffer significantly with respect to oxide

thickness and surface topography. The annealed

surface can be regarded as a smooth surface (on the

scale of cellular dimensions) with a thick surface

oxide, whereas the acid-oxidized surface is rough

even on a cellular size scale and has a thin surface

oxide. On the basis of previous work it is also safe to

assume that the microstructure and morphology ofthe oxides covering the two types of samples are

different, because these properties are strongly de-

pendent on oxidation conditionsY-3°

We can only speculate on the ultimate reason for

the different responses to the two titanium surfaces.

Possible factors that are involved are surface com-

position and structural properties (topography, ox-

ide thickness, and oxide microstructure). Because

the surface analyses indicated an essentially identi-

cal surface composition for the two different sur-

faces, we are left with the structural properties.

Several previous studies31-33 on other cell types haveshown that surface topography on the micron scale

and larger can dramatically influence the behavior

of cells on surfaces. However, our preliminary ex-

periments with titanium surfaces that are both

rough and have thick oxides indicate a protein ad-

sorption and cell adhesion/activation behavior sim-

ilar to that for the smooth surface with thick oxides.

These observations strongly indicate that the differ-

ences are mainly a the result of the microstructure

related to the oxide thickness. It is therefore appro-

priate to discuss some structural properties of tita-

nium oxide films and how they depend on surfacepreparation procedures.

It is well known that there is a close relationship

between the thickness and microstructure of oxide

films on titaniumY-3° Transmission electron mi-

croscopy studies29 of thin oxides have shown that

they are rather homogeneous and lack long-range

order (crystallinity). Thick oxides, prepared either

by thermal oxidation or by anodic oxidation, are

more heterogeneous, with areas of different poros-

ity. The size and distribution of these areas correlate

with the microstructure of the underlying metal sub-

strate. Oxides thicker than -40 nm also have a

higher degree of crystallinity, which increases with

thickness. Depending on the preparation method,

the oxides form the rutile or anastase forms of TiO2.

Rutile tends to be the dominant form obtained

after thermal oxidation at elevated temperatures

(>400 ° C). Annealing in air can also be expected to

result in oxide layers with fewer defects (e.g., a lower

conentration of oxygen vacancies). However, no sig-

nificant differences in oxygen defect densities were

indicated in the spectra from electron spectroscopy

for chemical analysis. Increasing oxide thickness can

also lead to increased built-in mechanical stress in

the surface film caused by the mismatch in the lat-

tice parameters and thermal properties between ti-

tanium and TiO2.

Thus the oxides covering the two titanium sur-

faces investigated here differ with respect to not

only the oxide thickness but also the microstructure,crystallinity, and built-in mechanical stresses, and

possibly also defect density. Most of these properties

can theoretically be expected to influence the initial

protein adsorption behavior (which in turn influ-

ences the cellular response). Oxide thickness per se

may influence protein adsorption behavior because

oxides of diffe rent thicknesses present different con-

ditions for electrostatic and van der Waals interac-

tions and also may result in differences in ion re-

lease rates. Microstructural properties and the

degree of crystallinity are also expected to influ-

ence molecular interactions at surfaces--for ex-ample, via differences in local interactions caused

by variations in electron band structure or via

steric effects. Defect densities are well known to

play a decisive role in the reactivity of oxide sur-

faces toward small molecules (e.g., H 2 0 ) and

should be expected to do so also in regard to

macromolecules, directly or via water interaction.

Defects may, for example, act as nucleation sites

for protein adsorption and are important for the

kinetics of protein adsorption at su rfacesY 36 It is

not possible from the present data to identify

which of these properties are the decisive factorsfor the different biologic responses observed.



J L a b C l in M e dV o l u m e 1 29 , N u m b e r 1 N y g r e n , E rik ss on , a n d L a u s m a a 4 5

T h e o b s e rv ed d i f f e r en ces i n t h e i n i t i a l l y fo rmed

p ro t e i n f i lm d o e f f ec t t h e ad h es i o n o f p la t e l e t s t o

t h e s u r f aces . A s u r f ace co v e rag e o f p la t e l e t s r e f l ec t-

i n g th e n o rma l i zed v o l u m e f r ac t i o n o f t h e ce l ls i n

b l o o d w o u l d c o r r e s p o n d t o 0 . 0 8 % ( c el l v o l u m e - 4 / 3

[ I r 3 X 400.000/mm3; r = 1.3 txm). Th e obs erv ed

s u r f ace co v e rag e a f t e r 5 s eco n d s o f b l o o d ex p o s u re

(3 % an d 6 % , r e s p ec t i v e l y ) t h u s r ep re s en t s ad h e r i n g

ce l l s , w i t h an i n c r eas ed r e s i d en ce t i me a t t h e s u r -

f ace . T h e o b s e rv e d d i f f e r en ce may b e ex p l a i n ed t o a

large ex ten t , bu t no t fu l ly , by the su r fac e en large-

m e n t f a c t o r ( 5 2 % ) .

E x p o s u r e o f C D 6 2 o n t h e s u r f a c e o f a d h e r i n g

p l a t e l e t s can b e s een a f t e r ap p ro x i ma t e l y 4 t o 8

mi n u t e s , i n d i ca t i n g t h a t t h e b i n d i n g t o ad h es i o n -

p ro mo t i n g p ro t e i n s i s n o t en o u g h t o ac t i v a t e t h e

cell s. Th i s f ind ing i s in cor re spo nde nce w i th those o f

o t h e r s . 9 Becau s e s ev e ra l ev en t s , i n c lu d i n g co m p l e -

men t ac t i v a t i o n , t h ro mb i n fo rma t i o n , an d f i b r i n o l y -

s is may have o ccu rred dur ing th i s t ime sp an , i t is no t

p o s s i b l e f ro m t h e p r e s e n t d a t a t o d e t e rm i n e t h e

cau s e o f CD 6 2 ex p re s s i o n .

A d h e s i o n o f P M N s c a n b e s e e n f r o m 8 t o 1 6

mi n u t e s , s h o w i n g t h a t p l a t e l e t s a r e ac t i v a t ed b e fo re

PMN s ad h e re t o t h e s u r f ace . T h e n o rma l i zed v o l -

u m e f r a c t io n o f P M N n u c l ei w o u l d c o r r e s p o n d t o

0 .0 6 % ( c e l l v o l u m e - 4 / 3 I I r 3 X 5000/mm3; r = 5

~xm). Th i s va lue i s no t rea che d un t i l 8 m inu tes o f

b l o o d ex p o s u re , i n d i ca t i n g t h a t t h e ce l l s d o n o t ad -here to the in i t i a l p ro te in l ayer , a l though PMN.s are

know n to car ry oL2131 and ~s [31 in teg r ins - - rec ep to rs

fo r adh es ion -pro mo t ing p ro te ins . 37-39 Th e cause o f

t h e h i g h e r co v e ra g e a t a la t e r t i me m ay b e a co m-

b i n a t i o n o f in c r eas ed r ec ru i t me n t b y ch emo t a~d s an d

ad h es i o n t o t h e s e l ec t i n ex p o s ed o n ac t i v a t ed p l a te -

le t s . 40-44 A l t e rn a t iv e l y , i t m ay b e c au s ed b y s o m e l a t e

mo di f i ca t ion o f the p ro te in f i lm, a l lowing adhe s ion

o f P M N s b y m e c h a n is m s u n k n o w n .

T h e d i f f e r en ces i n co mp o s i t i o n o f th e i n it ia l p ro -

t e i n f i l m , b e t w een t h e s u r f aces , may t h u s cau s e t h e

d i f f e r en ces i n ac t i v a ti o n o f p l a t e l e t s an d n eu t ro p h i l ss een mu ch l a t e r . I t may b e i mp o r t an t t o k eep i n

mi n d t h a t ev en t s in a b i o lo g i c r e s p o n s e i n t h e t i me

s ca le o f s eco n d s ma y ex p l a in e f f ec t s s een a f t e r h o u r s

o r p e rh ap s ev en d ay s . W e a r e p r e s en t l y fo l !o w i n g

the l ine o f even t s t ak ing p lace a t T iO2 sur faces in the

t i me s ca l e o f d ay s an d w eek s .

W e t h a n k A n n W e n n e r b e r g f o r p e r f o r m i n g t h e o p t i c a l p r o -

filometry analyses.

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