Proteins at Interfaces - American Chemical Society

groups replace surface phosphate i n the enamel hydroxy- apatite.(20,21) ... p r o t e i n o f s u b m a x i l l a r y s a l i v a (MW=12 χ 1 0 3 , 5...
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Chapter 27

Characterization of the Acquired Biofilms on Materials Exposed to Human Saliva H. J . Mueller

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Council on Dental Materials, American Dental Association, Chicago, IL 60611

A variety of dental alloys were submitted to adsorption experiments with human saliva. FT­ -IR and SIMS were used to analyze the surface films. IEF compared the protein patterns from surface extracts and salivas used in protein adsorptions to those from unexposed saliva controls. Results support both selective and nonselective adsorption processes. The SIMS spectrum showed v a r i a b i l i t i e s in elemental intensities between substrates of different compositions, while IEF patterns of surface extracts from eleven different compositions of powder a l l appeared to contain the same acidic protein bands. FT-IR spectrum showed v a r i a b i l i t i e s in the protein to carbohydrate intensity ratios at different sites on the same alloy surface, and suggested that other factors besides substrate material may be important in protein adsorption. S u r f a c e s c o m i n g i n t o c o n t a c t w i t h s a l i v a become a d s o r b e d i n a short time with a t h i n f i l m o f organic matter.(1-4) Much i n t e r e s t h a s b e e n g e n e r a t e d i n c h a r a c t e r i s i n g this f i l m f o r purposes o f e l u c i d a t i n g , ( i ) demineralizationm i n e r a l i z a t i o n processes o f enamel,(5) ( i i ) i n t e r a c t i o n s of enamel w i t h f l u o r i d a t i o n t r e a t m e n t s , ( 6 ) ( i i i ) i t s r o l e as p r e c u r s o r t o t h eattachment o f microorganisms and the f o r m a t i o n o f plaque, c a r i e s , and p e r i o d o n t a l disease (7), and i t s r o l e i n t a r n i s h i n g and c o r r o s i o n o f d e n t a l a l l o y s . ( 8 ) The r o l e o f adsorbed s a l i v a r y proteins, e s p e c i a l l y mucins, i n p r o t e c t i n g t h eo r a l t i s s u e s a g a i n s t environmental i n s u l t , p o t e n t i a l pathogens, and i n l u b r i ­ c a t i n g has been t a k e n a s r o u t i n e b i o l o g i c a l f u n c t i o n s . ( 9 ) S a l i v a - Enamel I n t e r a c t i o n s . Enamel becomes a d s o r b e d with a bacteria-free f i l m almost instantaneously a f t e r contacting s a l i v a , (10.) a n d i s c o n s t a n t l y renewable i f 0097-6156/87/0343-0435$06.00/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

PROTEINS AT INTERFACES

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l o s t o r abraded.(11) Langmuir's a d s o r p t i o n isotherm has b e e n u s e d w i t h some s u c c e s s e s p e c i a l l y a t l o w e r p r o t e i n concentrations i n following the adsorption.(12,13) Thick­ n e s s e s o f t h e o r d e r o f 1 0 - 2 0 nm a f t e r t h e f i r s t 1-2 h o u r s o f s a l i v a e x p o s u r e h a v e b e e n d e t e c t e d . ( 1-4 ) a l t h o u g h m u c h t h i c k e r f i l m s o f t h e o r d e r o f m i c r o n s have a l s o been d e m o n s t r a t e d . ( 1 1 ) The f i l m s i n c o n t r a s t t o enamel a r e a c i d i n s o l u b l e , although an a c i d s o l u b l e f r a c t i o n a l s o occurs,(14) and a c t as d i f f u s i o n b a r r i e r s a g a i n s t a c i d s . (IS) thereby r e d u c i n g t h e a c i d s o l u b i l i t y o f enamel and i n h i b i t i n g t h e a d h e r e n c e o f o r g a n i s m s . I t h a s become c u s t o m a r y t o r e f e r t o t h e i n i t i a l b a c t e r i a - f r e e integument, as t h e a c q u i r e d p e l l i c l e . ( 1 4 , 1 5 ) . Aged p e l l i c l e s c o n t a i n in a d d i t i o n t o t h e adsorbed p r o t e i n s , microorganisms, plaque, m i n e r a l i z e d products. and other debris.(7.11,16) A n a l y s i s o f e x t r a c t e d two h r i n - v i v o enamel p e l l i c l e showed i t t o be n e g a t i v e l y c h a r g e d and c o n t a i n i n g b o t h l o w e r a n d h i g h e r MW p r o t e i n s . G l y c i n e , g l u t a m i c a c i d , a n d s e r i n e were i n abundance and w i t h an amino a c i d c o n t e n t s i m i l a r t o a r e p o r t e d s a l i v a r y p h o s p h o p r o t e i n . (17.) I n addition, the p e l l i c l e s contain carbohydrate, which i n c l u d e s u p t o 70 % g l u c o s e ( 4 ) a n d l i p i d . ( 2 ) N e w i l - 2 h r ) p e l l i c l e s contain 30% of p r o l i n e - r i c h proteins. Their d e g r a d a t i o n b e g i n s a f t e r a b o u t 24 h r s . The p r o l i n e - r i c h p r o t e i n c o n t e n t i n a g e d p e l l i c l e s i s l e s s t h a n 0.1 % . ( 1 8 ) A n i o n i c d i s c g e le l e c t r o p h o r e s i s o f e x t r a c t e d two h r p e l l i c l e s i n d i c a t e d f o u r major bands and w i t h t h r e e bands i n d i c a t i n g m u l t i p l e sub-bands.(19) T h e a d s o r p t i o n o f s a l i v a r y p r o t e i n s t o e n a m e l may i n c l u d e exchange r e a c t i o n s i n which t h e p r o t e i n phosphate groups r e p l a c e s u r f a c e phosphate i n t h e enamel hydroxyapatite.(20,21) Salivary Binding Proteins. S a l i v a r y proteins that bind t o h y d r o x y a p a t i t e a n d w h i c h may b e i m p o r t a n t i n p e l l i c l e f o r m a t i o n i n c l u d e , ( 2 2 ) ( i ) m u c o u s g l y c o p r o t e i n s (MW=3-5 χ 1 0 , pl=2, 70% carbohydrate), ( i i ) proline-rich basic g l y c o p r o t e i n (MW=3.5 χ 1 0 , p l = 9 . 5 , 4 0 % C H O ) , ( i i i ) p r o l i n e - r i c h a c i d i c p r o t e i n (MW=6-12 χ 1 0 , p I = 4 - 4 . 7 ) , (iv) t y r o s i n e - r i c h p r o t e i n k n o w n a s s t a t h e r i n (MW=5.2 χ 1 0 , ρI = 4.2) , v ) h i s t i d i n e - r i c h p r o t e i n (MW=4.5 χ 1 0 , p l = 7 ) , a n d ( v i ) c a l c i u m g l y c o p r o t e i n o f m i x e d s a l i v a (MW= 6.2 χ 10-*, p l = 4.7, 1 5 % CHO). P r o t e i n s i n p a r t i i i , i v , and v i a l s o b i n d c a l c i u m . A c a l c i u m p r e c i p i t a b l e g l y c o ­ p r o t e i n o f s u b m a x i l l a r y s a l i v a (MW=12 χ 1 0 , 5 % C H O ) , w h i l e n o t b i n d i n g t o h y d r o x y a p a t i t e does bind c a l c i u m and is phosphorylated. P r o t e i n s i n p a r t s i i i , and ν a r e a l s o phosphorylated w h i l e t h e p r o t e i n s i n p a r t s i . and i i a r e sulfated. e

s

3

3

3

3

S a l i v a - Dental Materials Interactions. Besides enamel and o t h e r t i s s u e s , s u r f a c e s f r o m m e t a l l i c , p o l y m e r i c , a n d c e r a m i c d e n t a l m a t e r i a l s a r e c a p a b l e o f becomimg a d s o r b e d w i t h o r g a n i c f i l m s . Germanium and s i l i c a i n f r a r e d s p e c t r o ­ meter p r i s m s formed o r a l f i l m s a t h i g h speeds and were

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Characterization of the Acquired Biofilms

MUELLER

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s t a b l e over time. Detection of p r o t e i n , carbohydrate, and l i p i d was m a d e . ( 2 ) T h e a m i n o a c i d c o n t e n t o f f i l m s f o r m e d o n s e v e r a l p l a s t i c s a n d g l a s s v a r i e d a n d was d i f f e r e n t f r o m t h a t f o r m e d o n e n a m e l . I t was c o n c l u d e d t h a t t h e c h e m i c a l c o m p o s i t o i o n o f t h e s u b s t r a t e s u r f a c e has an important i n f l u e n c e on t h e t y p e o f p r o t e i n s w h i c h t h e y r e t a i n . ( 2 3 ) F o r p e l l i c l e s on d e n t u r e s , specific proteins s e e m e d t o b e p r e c u r s o r s i n f o r m i n g t h e f i l m s . (10.) F o r the p e l l i c l e s f r o m d i f f e r e n t r e s t o r a t i v e m a t e r i a l s , C, N, and Ο predominated the compositions. The t h i c k n e s s o f t h e f i l m f o r m e d o n g o l d a l l o y was a b o u t 10 x, 4 x , a n d 1.25 χ the t h i c k n e s s e s t h a t f o r m e d on amalgam, e n a m e l , and composite r e s i n , r e s p e c t i v e l y . No f i l m o c c u r r e d o n s i l i c a t e c e m e n t . T h e r e l e a s e o f F~ may h a v e c o m p e t e d f o r b i n d i n g s i t e s on t h e a n i o n i c a d s o r b i n g p r o t e i n s . C o p p e r was f o u n d i n f i l m o n g o l d a l l o y , w h i l e t i n was f o u n d i n f i l m o n a m a l g a m . T h e c a t i o n may be a f a c t o r i n t h e f o r m a t i o n and adhesion o f t h e f i l m s , and t h e a t t a c h m e n t o f m i c r o o r g a n i s m s . ( 3 ) B e s i d e s C, N, a n d O, t h e i n - v i v o t a r n i s h e d f i l m s o n g o l d a l l o y s c o n t a i n C l , S, C a , N i , Mg, S i , S n , F e , Κ, Na, Al a n d P. C o p p e r a n d Zn w e r e t h e o n l y a l l o y i n g e l e m e n t s i n the f i l m s . ( 8 ) Objective S a l i v a r y p r o t e i n s - d e n t a l m a t e r i a l s i n t e r a c t i o n s have not b e e n f u l l y a d d r e s s e d . The few i n number o f r e p o r t s h a v e been i n c o n c l u s i v e r e g a r d i n g i m p o r t a n t issues pertaining to adsorption. Further r e s u l t s are required to e l u c i d a t e i n g r e a t e r d e t a i l the a d s o r p t i o n of s a l i v a r y components t o d e n t a l m a t e r i a l s s u r f a c e s . Whether the adsorption p r o c e s s e s on a l l s u r f a c e s a r e s p e c i f i c t o a few proteins or whether the adsorbed p r o t e i n s depend upon the under­ l y i n g substrate composition i s v e r y much o f I n t e r e s t . I t was t h e g o a l o f t h i s p r o j e c t t o i n v e s t i g a t e t h e e f f e c t of d e n t a l m a t e r i a l c o m p o s i t i o n upon the adsorbed f i l m c h a r a c t e r i s t i c s . What e l e m e n t s b e c o m e a d s o r b e d and what compounds a r e formed or a d s o r b e d were i n v e s t i g a t e d . I n c l u d e d was a c o m p a r i s o n o f t h e a d s o r b e d p r o t e i n s t o those o c c u r r i n g i n s a l i v a . Since the f i l m s are u s u a l l y o n l y nm i n t h i c k n e s s , t h e a p p r o p r i a t e a n a l y t i c a l m e t h o d s w e r e r e q u i r e d f o r t h e i r a n a l y s i s . I t was t h e p u r p o s e o f t h i s poroject to u t i l i z e both f o u r i e r transform i n f r a r e d s p e c t r o m e t r y ( F T - I R ) and s e c o n d a r y i o n mass s p e c t r o m e t r y ( S I M S ) , two h i g h l y s u r f a c e o r i e n t a t e d t e c h n i q u e s , as w e l l as i s o e l e c t r i c f o c u s i n g . FT-IR i s c a p a b l e o f d e t e c t i n g o r g a n i c s t r u c t u r e s , w h i l e SIMS i s c a p a b l e o f d e t e c t i n g most o f t h e e l e m e n t s w i t h i n s e v e r a l o f t h e o u t e r m o s t m o n o l a y e r s . I s o e l e c t r i c f o c u s i n g i s an e l e c t r o p h o r e t i c t e c h n i q u e made i n pH g r a d i e n t . T h e r e s u l t i n g b a n d s a l o n g t h e pH g r a d i e n t c o r r e s p o n d t o t h e i s o e l e c t r i c p o i n t s o f included proteins. Materials Saliva

and

and

Methods

A l l o y Surface

Preparations.

Unstimulated

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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PROTEINS AT INTERFACES

w h o l e s a l i v a was c o l l e c t e d i n t o i c e c h i l l e d p o l y p r o p l y e n e b e a k e r s f r o m one d o n o r a t t h e b e g i n n i n g o f e a c h o f t h e d i f f e r e n t e x p e r i m e n t s . The s a m p l e s w e r e c e n t r i f u s e d i n p o l y p r o p l y e n e t u b e s a t 1 6 0 0 >: g f o r 30 m i n u t e s . T h e s u p e r ­ n a t a n t s were d e c a n t e d and u s e d i n a d s o r p t i o n t e s t s w i t h v a r i o u s a l l o y s u r f a c e s o r p o w d e r s t o be d e s c r i b e d . A l l o v s u r f a c e s f o r F T - I R and SIMS were i n i t i a l l y g r o u n d t o a no. 600 g r i t f i n i s h on s i l i c o n c a r b i d e s t r i p s , f o l l o w e d by 10 urn a n d 5 urn g r i n d i n g o n S t r u e r ' s r o t a t i n g S i C d i s c s . The s u r f a c e s w e r e p o l i s h e d w i t h 3 and 1 urn d i a m o n d p a s t e s o n n a p c l o t h s . S a m p l e s w e r e i m m e r s e d u l t r a s o n i c a l l y i n d e t e r e g e n t , d e i o n i z e d w a t e r r i n s e d , and d e c r e a s e d by i m m e r s i o n s i n a c e t o n e f o l l o w e d b y C C I * . F o u r i e r Transform I n f r a r e d Spectrometry (F^-IR) Four c r o w n a n d b r i d g e a l l o y s , a l l o y s A, B. C, a n d Ε i n T a b l e Table

I. A l l o v

Surfaces Analyzed

Allov Manufacturer A Szabo Heraeus Β Midas Jelenko C A 1 b a c a st J e l e n k o D Tytin S.S. White Ε MS Monarch F Biobond Dentsplv

by

FT-IR

or

I,

SIMS

Composition(wt %) other Cu Au Pd Α** 77 . 4 8 .1 12. 5 2 7 .C 39. c ^6 . 0 6 — 25 5 .0 70 . 0 7 .c 16Sn, 42Hg 34 . c _ Fe.Ni,Mn 72 . 0 20A1 Sn.Nb.Si.B b a l 7 6 N i , 1 2 C r . 3Mo (

-

-

(

f

mm thick. w e r e c a s t i n t o s q u a r e s h a p e s 8 Χ­ S mm a n d 1 .5 The p o l i s h e d s a m p l e s w e r e e x p o s e d f o r a few d a y s t o t h e o r a l e n v i r o n m e n t . The s a m p l e s w i t h h o l e s d r i l l e d length­ w i s e were a t t a c h e d v i a o r t h o d o n t i c e l a s t i c t h r e a d t o p l a s t i c b r a c k e t s cemented to the b u c c a l s u r f a c e s of upper molar or b i c u s p i d teeth.(24) A f t e r removal, the samples were r i n s e d w i t h d i s t i l l e d H Q and a i r d r y e d . The s a m p l e s w e r e a n a l y z e d i n t h e r e f l e c t i v e mode o n a D i g i l a b F T - I R . I n a n o t h e r s e r i e s , g o l d a l l o y A ( T a b l e I ) was exposed i n - v i t r o f o r 1 hr to the supernatant of s a l i v a . A f t e r d i s t i l l e d w a t e r r i n s i n g a n d a i r d r y i n g , t h e s u r f a c e was v i e w e d u n d e r t h e FT-IR m i c r o s c o p e and s u r f a c e s c r a p i n g s made w i t h a s c a p e l t i p a t d i f f e r e n t l o c a t i o n s a c r o s s t h e s u r f a c e . O r g a n i c m a t e r i a l removed a t t h e d i f f e r e n t s i t e s was o b t a i n e d a s t h i n f i l m s o n ΚBr d i s c s . An A n a l e c t F T IR was u s e d t o o b t a i n t h e s p e c t r u m 2

S e c o n d a r y Ion Mass S p e c t r o m e t r y . P o l i s h e d a l l o y s , A, C, D. E. a n d F i n T a b l e I w e r e e x p o s e d t o t h e s u p e r n a t a n t o f human s a l i v a , w a t e r r i n s e d , a i r d r y e d , a n d a n a l y z e d b y SIMS. A C a m b r i d g e S t e r e o s c a n 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 e w i t h a n a t t a c h e d a r g o n beam g u n a n d q u a d r a p o l e m a s s a n a l y z e r ( K r a t o s S I M S u n i t ) was u s e d . B o t h t h e p o s i t i v e and n e g a t i v e s p e c t r u m were t a k e n . A f t e r SIMS a n a l y s i s , t h e s a m p l e s w e r e r e g r o u n d and r e p o l i s h e d and a n a l y z e d a g a i n t o o b t a i n SIMS s p e c t r u m o f t h e a s - p o l i s h e d s u r f a c e s . A d s o r p t i o n Methods f o r I s o e l e c t r i c F o c u s i n g . F i v e ml

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

27. MUELLER

439

Characterization of the Acquired Biofilms

o f t h e s u p e r n a t a n t w e r e m i x e d w i t h 5 0 mg o f d i f f e r e n t d e n t a l m a t e r i a l s powders c o n t a i n e d i n p o l y s t y r e n e tubes. Table I I c h a r a c t e r i z e s t h e powders. I n i t i a l l y t h e tube

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Table

1 ο 3 4 5 6 7 8 9 10 11

I I .Alloy

Powder Human E n a m e l Hydroxyapatite Porcelain PMMA r e s i n Palladium Silver Ag-Cu e u t e c t i c Copper Tin Amalgam A l l o y Bismuth

Powders f o r P r o t e i n A d s o r p t i o n Source ground t o o t h Sigma Dentsply General Dental Alfa Goldsmith Consolidated Astro Sargent-Welch Fisher Engelhard Goldsmith

with IEF

Particle Size -200 mesh -200 mesh -200 mesh -150 mesh 0 . 2 5 - 0 . 5 5 urn -325 mesh -325 mesh -150 mesh -325 mesh -325 mesh -325 mesh

c o n t e n t s were v i g o r o u s l y shaken w i t h tube mixer f o l l o w e d by a 4 h r i n c u b a t i o n w i t h a m o d e r a t e l i n e a r b a c k a n d f o r t h m o t i o n . The powders were s e p a r a t e d from t h e s a l i v a by c e n t r i f u g i n g a t 1 6 0 0 x g f o r 15 m i n u t e s . T h e p o w d e r s w e r e washed w i t h 5 ml o f d i s t i l l e d H O by v i g o r o u s l y s h a k i n g f o r 5 m i n w i t h t u b e m i x e r and c o l l e c t e d by c e n t r i f u g i n g . Two e x t r a c t i o n s f o l l o w e d , t h e f i r s t w i t h 0.2 M N a H P C u a n d t h e s e c o n d w i t h 0.2 M E D T A ( N a ^ ) . F i v e m l o f t h e e x t r a c t i o n s o l u t i o n were added t o each tube, v i g o r o u s l y s h a k e n f o r s e v e r a l m i n u t e s and f o l l o w e d by an i n c u b a t i o n f o r 4 h r . The e x t r a c t s were c o l l e c t e d by c e n t r i f u g i n g , d i a l y z e d f o r 24 h r s a g a i n s t d i s t i l l e d H o w i t h 1000 MWCO m e m b r a n e s . C o n c e n t r a t i o n f o l l w e d b y d e h y d r a t i o n to near d r y n e s s i n a 3 5 % s o l u t i o n o f p o l y e t h y l e n e g l y c o l . The s o l u t i o i n s w e r e r e c o n s t i t u t e d b y a d d e d 0.15 m l o f d i s t i l l e d water. s

2

2

P o l y a c r y l a m i d e and Agarose I s o e l e c t r i c F o c u s i n g IEF was p e r f o r m e d o n a L K B M u l t i p h o r 2 1 1 7 e l e c t r o p h o r e s i s u n i t with e i t h e r polyacrylamide or agarase g e l p l a t e s which contained ampholine c a r r i e r ampholytes f o rg e n e r a t i n g t h e pH g r a d i e n t 3.5 t o 9 . 5 . W i t h t h e 1 mm t h i c k p o l y a c r y l a m i d e p r e c a s t g e l p l a t e s , 1 M H^PO* a n d 1M NaOH w e r e a d d e d t o t h e a n o d e a n d c a t h o d e , r e s p e c t i v e l y , w h i l e w i t h t h e 0.5 mm t h i c k c a s t a g a r o s e p l a t e s a 0.5 M a c e t i c a c i d a n d 0.5 M NaOH w e r e u s e d . C o o l i n g o f t h e g e l s w a s b y 5 d e g C w a t e r flowing i n t h e cooling plate. In focusing a p l a t e having d i m e n s i o n s o f 1 1 0 χ 2 4 5 mm, a c o n s t a n t p o w e r o f 2 0 W w a s a p p l i e d f o r 1 1/2 h r s w i t h t h e a c r y l a m i d e g e l s a n d f o r 1/2 h r w i t h t h e a g a r o s e g e l s . T h e pH g r a d i e n t a c r o s s t h e g e l w i d t h s was m e a s u r e d w i t h a n LKB s u r f a c e c o m b i n a t i o n pH e l e c t r o d e . The g e l s were f i x e d i n a 11.5% t r i c h l o r o a c e t i c a c i d - 3.5% s u l p h o s a l i c y l i c a c i d s o l u t i o n and r i n s e d i n 95% e t h a n o l t o remove a m p h o l i n e . The p o l y a c r y l a m i d e gels were s t a i n e d w i t h a s o l u t i o n c o n t a i n i n g 1.15% c o o m a s s i e b l u e R 2 5 0 , 8% a c e t i c a c i d , a n d 2 5 % e t h a n o l s o l u t i o n .

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

440

PROTEINS AT INTERFACES

D e s t a i n i n s was w i t h s t a i n i n g s o l u t i o n w i t h o u t a d d e d c o o m a s s i e b l u e . The a g a r o s e p l a t e s were s t a i n e d w i t h s i l v e r (25) A f t e r r e m o v i n g a m p h o l i n e and d r y i n g , t h e g e l s were i m m e r s e d i n a 2% KFeCN f o r 5 min, r i n s e d i n w a t e r , a n d s t a i n e d f o r 15 m i n i n d e v e l o p i n g s o l u t i o n w h i c h c o n t a i n e d 35 % o f s o l u t i o n A a n d 6 5 % o f s o l u t i o n Β. S o l u t i o n A w a s c o m p o s e d o f 8% N a C O , w h i l e s o l u t i o n Β w a s c o m p o s e d o f 0 . 1 9 % o f NH^NO.3, 0 . 2 % A g N 0 , 1 % t u n g s t o s i l i c i c a c i d , and 7 . 3 % ( v / v ) f o r m a l i n ( 3 7 % ) . 2

3

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3

Results FT-IR. F i g u r e 1 p r e s e n t s an FT-IR s p e c t r a t a k e n from the surface of a l l o y A i n Table 1 a f t e r i n - v i v o exposure. The a m i d e I a n d I I p r o t e i n b a n d s a r e d e t e c t e d a t a b o u t 1650 c m and 1530 cm" , r e s p e c t i v e l y , as w e l l as a d d i t i o n a l p r o t e i n bands a t 1450 c m and 1390 cm" . S t r o n g c a r b o h y d r a t e a d s o r p t i o n i s d e t e c t e d a t 1060 c m , as w e l l a s m o d e r a t e l i p i d c o n t e n t a t 1250 c n r . A d d i t i o n a l a d s o r p t i o n peaks i n c l u d e C H a t about 2930 cm" and C 0 a t 2 3 5 0 c m . S p e c t r u m f r o m t h e r e m a i n i n g a l l o y s (Β, C, Ε i n T a b l e I ) w e r e s i m i l a r t o t h a t f r o m a l l o y A. T h e u s e o f d e c o n v o l u t i o n methods f a i l e d t o produce any s i g n i f i c a n t d i f f e r e n c e s due t o a l l o v c o m p o s i t i o n . The s p e c t r u m f r o m t h e s c r a p i n g s f o r d i f f e r e n t s i t e s on t h e s u r f a c e o f a l l o y A a f t e r o n l y 1 h r o f i n - v i t r o e x p o s u r e s t o t h e s u p e r n a t a n t o f s a l i v a showed v a r i e d r e s u l t s . A comparison of t h e r a t i o s o f t h e p r o t e i n ( o r l i p i d ) t o c a r b o h y d r a t e peak i n t e n s i t i e s a t f o u r d i f f e r e n t surface s i t e s i spresented i n Table I I I . - 1

1

- 1

1

- 1

1

1

3

2

- 1

Table

I I I . P r o t e i n ( o r L i p i d ) t o C a r b o h y d r a t e Peak R a t i o s (% T/% T) a t F o u r S u r f a c e S i t e s o n A l l o y A Site

Peak(cm

- 1

>

1 77 .. 9 3 .. I l l .. 112. . 114 ..

1650 1530 1450 1390 1250

9 1 0 0 0

3 77. , 8 2 .. 98. . 110 .. 1 0 2 ..

2 65. . 5 76. . 1 96. , 0 9 6 ..2 98 .. 8

2 2 5 0 0

4 74 .. 4 ο / ., 2 102. , 0 104 .. 0 105. c

SIMS. F i g u r e 2 p r e s e n t s a " p o s i t i v e " SIMS s p e c t r a f o r a l l o y A a f t e r s a l i v a exposure. Comparison t o t h e spectrum from d i f f e r e n t a l l o y s i n d i c a t e d t h e f o l l o w i n g . The peaks r e l a t e d t o a l l o y i n g e l e m e n t s a r e Cu(AMU=63 & 6 5 ) f o r a l l o y s A, C, D, a n d E, A l < AMU=27) , M n ( A M U = 5 6 ) , a n d F e ( A M U = 5 4 , 5 6 , 5 7 , 5 8 ) f o r a l l o y Ε. a n d B ( A M U = 1 1 ) , S i ( A M U = 2 8 , 2 9 , 3 0 ) . C r ( A M U = 5 0 , 5 2 , 5 3 , 5 4 ) a n d N b ( A M U = 9 3 ) f o r a l l o y F. N i c k e l ( A M U = 5 8 , 6 0 , 6 1 . 6 2 , 6 4 ) o c c u r s w i t h a l l o y s Ε a n d F. O t h e r s t r o n g p e a k s i n c l u d e Na ( A M U = 2 3 ) , Κ ( A M U = 3 9 , 4 1 ) , a n d C a ( A M U = 4 0 . 4 2 , 4 3 , 4 4 ) . P e a k s a l s o o c c u r a t AMU o f 1 2 , 1 3 , 14 ( Ν ) , 1 5 Î N H ) , 16, 2 7 Î C N H , C H , o r A l ) . a n d 4 3 ( C H Q ) . P e a k s a l s o o c c u r f o r CrO(AMU=68), NiO(AMU=74), a n d CuO(AMU=80). T a b l e s I V a n d V p r e s e n t c o m p a r i s o n s b e t w e e n SIMS peak 2

3

2

3

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Characterization of the Acquired Biofilms

MUELLER

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1.00

^

w

4000

ι

1

1

1

1

1

1

3600

3200

2800

2400

2000

1600

1200

800

WAVE NUMBERS

F i g u r e 1. FT-IR s p e c t r a f r o m s u r f a c e o f a l l o y A w h i c h was r e t r i e v e d from i n v i v o u s a g e .

Ο

10

20

30

40

50

60

70

A T O M I C M A S S UNITS (AMU)

F i g u r e 2. P o s i t i v e SIMS s p e c t r a f r o m s u r f a c e o f a l l o y A w h i c h was e x p o s e d t o t h e s u p e r n a t a n t o f human s a l i v a f o r s e v e r a l h o u r s .

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

PROTEINS AT INTERFACES

442

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Table .

AMU 11 12 13 14 15 16 23 28 39 40 43 52 55 56 58 63 68 74 93 107-10 116-22 Table

IV. P o s i t i v e SIMS Peak m i n u s B a c k g r o u n d Intensities (counts) f o r S a l i v a Exposed A l l o v Surfaces

A

C

10

c— 19 48

48

1714 123 58 666 571

28 1666 57 514 209

Allov D 76 57 104 20 10 171 28 28 304 542

Ε 133 104 209 228 48 152 476 266 1856 1808 76

F 209 19 133 86 95 1332 190 352 323 238 1475

57

95

104

48 48 19 1428

875 67 38 57

3 7 , 28

V.

84 , 7 5

28 56

P o s i t i v e SIMS Peak minus B a c k g r o u n d I n t e n s i t i e s ( c o u n t s ) f o r As-- P o l i s h e d A l l o v S u r f a c e s

AMU A 11 12 113 13 113 14 283 15 368 57 16 23 2518 27 452 28 113 39 509 40 226 43 52 55 198 56 141 58 63 3537 68 74 93 107-10 680,481 116-22

C

Alloy D

Ε

5377 622 141 453 233 57

28 28 226 226 56 4245 1201 198 877 538 226

85 56 141 198 28 4811 6226

198 170

396 368

651

1068

1641 1584 452 5094

85 198 311

962 113 141

85 85 3962,3900 4000

339,311 1075

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

F 283

113 141 28 6226 1641 1198 850 1301 95 5377

3962 849 283 198

27.

MUELLER

Characterization of the Acquired Biofilms

i n t e n s i t i e s f o r s a l i v a exposed and a s - p o l i s h e d s u r f a c e s , r e s p e c t i v e l y . P e a k s w i t h a t o m i c mass u n i t s h i g h e r t h a n t h o s e s h o w n i n F i g u r e 2 i n c l u d e Ag(AMU= 1 0 7 , 1 0 9 ) , P d ( A M U = 1 0 4 , 5 , 6 , 8 , 1 0 ) , SnfAMU=116-20,22,24), and SnO(AMU=132-40). A l l f i v e o f t h e a s - p o l i s h e d s u r f a c e s e x h i b i t e d h i g h e r Na i n t e n s i t i e s a n d h i g h e r i n t e n s i t i e s f o r AMU= 2 7 (C-H-,. CNH. or A l ) and 4 3 C C H Q ) . The a l l o y i n g e l e m e n t s c o n t a i n e d in t h e substrate a r e also higher i n i n t e n s i t y f o r t h e asp o l i s h e d s u r f a c e s . This i s c l e a r l y e v i d e n t i n Tables IV f o r C r , Mn, F e , N i , C u . Nb. A g , P d , a n d S n , a s w e l l a s f o r C r O , N i O , C u O , a n d SnO. P e a k s a l s o o c c u r r e d w i t h f o u r o u t o f t h e f i v e a l l o y s a t AMU = 5 5 a n d 5 6 . F o r a l l o y Ε t h e s e a r e f r o m Mn a n d F e , w h i l e f o r t h e o t h e r s , C a O i s i n v o l v e d . F i g u r e 3 p r e s e n t s t h e " n e g a t i v e " SIMS s p e c t r a f o r a l l o y A i n t h e s a l i v a exposed c o n d i t i o n . Peak i n t e n s i t i e s f o r C(AMU=12), CH(AMU=13), 0(AMU=16). 0H(AMU=17). F(AMU = 1 9 ) , a n d CN(AMU=26 o r C H ) a r e h i g h e r f o r t h e e x p o s e d c o n d i t i o n . H o w e v e r , t h e C I ( A M U = 3 5 , 3 7 ) p e a k was higher i n t h e as-polished condition. S i m i l a r trends t o o k p l a c e w i t h a l l o y s C a n d F, w h i l e a l l o y s D a n d Ε h a d higher Ο and C I i n t e n s i t i e s w i t h t h e s a l i v a exposed s t a t e . T a b l e s VI and V I I p r e s e n t c o m p a r i s o n s o f t h e v a r i o u s peak i n t e n s i t i e s f o r both s a l i v a exposed and a s - p o l i s h e d s t a t e s . 2

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443

3

2

2

T a b l e V I ,. N e g a t i v e S I M S P e a k m i n u s B a c k g r o u n d I n t e n s i t i e s (counts) f o r S a l i v a Exposed A l l o y s

( AMU ) 12 13 16 17 19 24 25 26 32 c

,

A 3966 5477 10953 3399 3021 661 472 2455 566 2266

C 5476 7365 11993 3871 3305 1133 755 3116 944 3772

T a b l e V I I . N e g a t i v e SIMS (counts)

( AMU ) 12 13 16 17 19 24 25 26 32 35

A 906 1528 3679 906 1415 452 340 1358 339 3906

C 708 1443 3226 934 3113 340 283 1075 452 4528

Alloy D 1019 1301 4160 1075 3679 283 141 509 378 907

Ε 1952 1840 2009 707 2066 509

F 1322 2172 12842 7460 566

1211

94 378 236

-

424 509

Peak minus Background I n t e n s i t i e s f o r As-Polished Alloys Allov D 1726 2377 5264

Ε 1132 2037 5716

F 962 1726 5150

3339 339 226 311 481 5086

4358 2830 2264 2264 396 4245

2575 85 113 85 339 2094

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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444

PROTEINS AT INTERFACES

10

20

30

40

50

60

A T O M I C M A S S U N I T S (AMU)

F i g u r e 3. N e g a t i v e SIMS s p e c t r a from s u r f a c e o f a l l o y A w h i c h was e x p o s e d t o t h e s u p e r n a t a n t o f human s a l i v a f o r s e v e r a l h o u r s .

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

27.

MUELLER

445

Characterization of the Acquired Biofilms

T a b l e V I I I p r e s e n t s t h e change i n peak i n t e n s i t i e s minus b a c k g r o u n d f o r a l l o y A e x p o s e d t o s a l i v a a f t e r a 4 h r a r g o n i o n beam e t c h .

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Table

V I I I . P e r c e n t Change i n SIMS S p e c t r u m f o r ,S a l i v a E x p o s e d A l l o y A a f t e r A r g o n Ion Beam E t c h

14 -40

16 -50

17 -99

P o s i t i v e SIMS(AMU) 18 23 27 -99 +30 +667

28 -87

39 + 17

40 + 113

12 -38

13 -18

16 -13

N e g a t i v e SIMS(AMU) 17 19 24 -49 +17 -99

26 -14

32 -50

"35 + 50

IEF. F i g u r e 4 presents a photogrraph of polyacrylamide g e l p l a t e s t a i n e d with coomassie blue f o r s a l i v a supern a t a n t s t h a t were i n c o n t a c t w i t h t h e e l e v e n powders i n t h e a d s o r p t i o n e x p e r i m e n t s . The t w e l f t h p a t t e r n i s f o r an u n e x p o s e d s a l i v a c o n t r o l sample. A l l p a t t e r n s a p p e a r e d s i m i l a r and as many as 2 5 p r o t e i n bands were d i s c e r n a b l e by i n s p e c t i o n . However, o n l y a l i m i t e d number were e a s i l y d e t e c t a b l e by o b s e r v a t i o n . P r o t e i n s were d e t e c t e d w i t h i n t h e pH r a n g e o f 4.4 t o 8.9. The p r o t e i n s o c c u r r i n g between pH = 5.2 - 5.7 and a t 6.6, 6.8, and 7.2 were most i n t e n s e and d i s c e r n a b l e . A s c h m e t a t i c r e p r e s e n t a t i o n o f t h e p a t t e r n s and t h e pH g r a d i e n t c o r r e s p o n d i n g t o t h e p l a t e d i m e n s i o n s a r e shown i n F i g u r e 5. The IEF p a t t e r n s f o r t h e H PCU and EDTA e x t r a c t s from t h e e l e v e n powders were v e r y weak i n i n t e n s i t y . The p a t t e r n s from t h e enamel and h y d r o x a p a t i t e e x t r a c t s , e v e n t h o u g h j u s t b a r e l y d i s c e r n a b l e , were s t i l l t h e most i n t e n s e from a l l o f t h e e x t r a c t s . T h e s e p a t t e r n s i n d i c a t e d s e v e r a l p r o t e i n l i n e s a t t h e same p o s i t i o n s as o c c u r r i n g w i t h t h e t h e c o n t r o l and s u p e r n a t a n t s . T h e s e l i n e s c o r r e s ponded t o t h e pH = 5-6 r a n g e and c l o s e t o 7. A l l r e m a i n i n g e x t r a c t s o l u t i o n s o n l y gave v e r y weak i n d i c a t i o n s f o r t h e e x i s t e n c e o f s e v e r a l bands and w i t h i n t h e pH = 5-6 r a n g e . S c h e m a t i c s f o r t h e s e p a t t e r n s a r e a l s o shown i n F i g u r e 5. 3

Discussion Some o f t h e d a t a from t h i s p r o j e c t s u p p o r t s a s e l e c t i v e a d s o r p t i o n p r o c e s s , w h i l e some o t h e r d a t a t e n d s t o s u p p o r t t h e o p p o s i t e v i e w p o i n t . The d a t a from t h e SIMS work shows v a r i a b i l i t y i n t h e s u r f a c e c o m p o s i t i o n s among t h e v a r i o u s s u b s t r a t e m a t e r i a l s , a l t h o u g h some t r e n d s were a l s o s e e n . The d a t a from t h e IEF a n a l y s e s i n d i c a t e d non s p e c i f i c a d s o r p t i o n , s i n c e t h e same p r o t e i n s a p p e a r e d t o be bound t o a l l o f t h e d i f f e r e n t s u b s t r a t e c o m p o s i t i o n s u s e d . The FT-IR r e s u l t s showed t h a t d i f f e r e n c e s i n a d s o r b e d f i l m c o m p o s i t i o n can o c c u r e v e n on t h e same s u b s t r a t e m a t e r i a l , and s u g g e s t e d t h e p o s s i b i l i t i e s f o r o t h e r f a c t o r s b e s i d e s s u b s t r a t e c o m p o s i t i o n t o be i m p o r t a n t i n a d s o r p t i o n . Some o f t h e s e o t h e r f a c t o r s may i n c l u d e s u r f a c e s m o o t h n e s s and preparation.

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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446

PROTEINS AT INTERFACES

Ό

U3

Ο

α J2 υ

α *~ oo w

00 C

c

t-

*s O

C3

-C

eu

OO oo

α ι

!& S3 KJ

O

o ^ α > 00

o c

-si

g> o S o v* υ

S& oo

ε

MU oo

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

27.

MUELLER

Characterization of the Acquired Biofilms

447

WIDTH (CM)

0 I

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4

2 1

1

1

6 1

I

8 I

I

10 12 I

I

l _

Φ­

ο ιI I

σο >

F i g u r e 5. G r a d i e n t i n pH d e v e l o p e d a c r o s s p l a t e s h o w n i n F i g u r e 4. S c h e m a t i c s A, Β. a n d C r e f e r t o patterns f o r s a l i v a c o n t r o l , phosphate e x t r a c t f r o m e n a m e l o r h y d r o x y a p a t i t e . a n d EDTA e x t r a c t s from a l l powders.

The e f f e c t s f r o m s h o r t t i m e s a l i v a e x p o s u r e s u p o n s u r f a c e c h a r a c t e r i s t i c s o f t h e a l l o y s a r e e v i d e n t from t h e SIMS s p e c t r u m . T h i s i s most c l e a r l y s e e n b y t h e l o w e r i n g i n t h e a l l o y i n g element c h a r a c t e r i s t i c i n t e n s i t i e s . This most d e f i n i t e l y e s t a b l i s h e s a n a d s o r b e d l a y e r o r f i l m o f some k i n d . T h e c h a r a c t e r i s t i c s o f t h e a d s o r b e d f i l m s included v a r i a b i l i t i e s i norganics aswell as inorganic c o n t e n t s . The v a r i a t i o n s i n o r g a n i c c o n t e n t s a r e seen i n t h e p o s i t i v e S I M S d a t a b y AMU = 2 7 ( C N H o r C H } a n d 43 (C-H.^G) a n d i n t h e n e g a t i v e S I M S d a t a b y AMU = 1 2 ( C ) , 13(CH). 16(0), 17(0H), 2 4 ( C ) , and 26(CN). C e r t a i n l y t h e v a r i a t i o n s i n some o f t h e s e s p e c i e s w i t h s u b s t r a t e material supports s e l e c t i v e adsorption. For t h e inorganic" c o n t e n t s , t h e h i g h l e v e l s o f N a , K. a n d C a n o t o n l y i n t h e outermost t o p surface l a y e r s , but also t h e i r increased c o n t e n t s w i t h f i l m d e p t h ( a r g o n e t c h i n g t i m e ) may p r o v i d e e l e c t r i c a l c o n d u c t i v i t y pathways through t h e f l i m t o t h e s u b s t r a t e . The r e l e a s e o f s u b s t r a t e i o n s v i a c o r r o s i o n may t h e n a f f e c t t h e a d s o r p t i o n p r o c e s s e s o f f i l m o n t o s u b s t r a t e a n d h e n c e make a d s o p t i o n a s e l e c t i v e p r o c e s s . T h e s e e f f e c t s may b e a c c e n t u a t e d o r m i n i m i z e d by t h e v a r i a b i l i t i e s i n t h e c o n c e n t r a t i o n s o f N a , Κ, a n d C a w i t h s u b s t r a t e a l l o y . The v a r i a t i o n s i n C I w i t h s u b s t r a t e and i t s i n c r e a s e d c o n c e n t r a t i o n w i t h f i l m t h i c k n e s s a r e also l i k e l y to affect theadosorption processes. The h i g h l e v e l s o f i o n s r e m a i n i n g o n t h e s u r f a c e s 2

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after preparations (as-polished condition) i s also very l i k e l y t o affect the adsorption processes. For a l l f i v e a l l o y s , t h e C I c o n c e n t r a t i o n on t h e a s - p o l i s h e d s u r f a c e s w e r e v e r y much h i g h e r t h a n o n t h e s u r f a c e s a f t e r s a l i v a e x p o s u r e s . T h i s r e d u c t i o n i n o u t e r m o s t C I may b e p a r t l y due t o r e l e a s e o f C I i n t o s o l u t i o n , o r d i f f u s i o n and penetration further into the film thickness. Also, the h i g h e r l e v e l s o f o r g a n i c s r e t a i n e d on t h e a s - p o l i s h e d surfaces suggest that the s t a t e of the as-polished surface may h a v e m o r e o f a n i n f l u e n c e t h a n t h e a c t u a l s u b s t r a t e on t h e a d s o r p t i o n p r o c e s s e s . S i n c e t h e t o p s u r f a c e l a y e r s i n t e r a c t w i t h t h e p r o t e i n s a n d i o n s i n s a l i v a , i t may b e t h a t t h e s e t o p l a y e r s o f i o n s may b e c o n t r o l l i n g processes r e l a t e d t o a d s o r p t i o n . When v i e w e d o n t h e a t o m i s t i c s c a l e t h e a d s o r p t i o n o f s a l i v a r y p r o t e i n s may j u s t b e a r e m o d i ­ f i c a t i o n of the outermost t o p surface l a y e r s , already very c o m p l e x e v e n b e f o r e p r o t e i n c o n t a c t . One t h e n s h o u l d t h e n be c o n c e r n e d w i t h s u r f a c e a l l o y p r e p a r a t i o n s i n s t e a d o f the a c t u a l s u b s t r a t e m a t e r i a l . The i n a b i l i t y t o u n e q u i v o c a l l y e s t a b l i s h d i s t i n c t and i n t e n s e IEF p a t t e r n s f o r t h e e x t r a c t s from t h e v a r i o u s powders remained a f t e r i n t e n s i f i e d measures f o r b e t t e r d e f i n i t i o n s . A number o f a v a i l a b l e m e t h o d s w e r e u s e d t h a t a r e k n o w n t o i n f l u e n c e I E F p a t t e r n s . Some a d d i t i o n a l e f f o r t s u n d e r t a k e n i n c l u d e d e x t r a c t d i a l y s i s and concen­ t r a t i o n prior t o IEF. Besides, i n applying the extracts to t h e g e l p l a t e s , n o t one b u t f o u r and more a p p l i c a t i o n s t r i p s were a p p l i e d t o t h e g e l s , thus p e r m i t t i n g a t l e a s t f o u r t i m e s t h e amount o f e x t r a c t t o be a p p l i e d . The u s e of agarose instead of polyacrylamide gels permitted the very highly s e n s i t i v e s i l v e r s t a i n i n g technique(25) t o be used. However, even h e r e t h e r e s u l t i n g p a t t e r n s f o r t h e e x t r a c t s l a c k e d d e f i n i t i o n and c l a r i t y which i s r e q u i r e d f o r p h o t o g r a p h y e x h i b i t i o n . The s i m i l a r i t i e s i n f e a t u r e s f r o m a l l t h e I E F e x t r a c t p a t t e r n s s u g g e s t e d t h e same s a l i v a r y p r o t e i n s , w i t h i s o e l e c t r i c p o i n t s a r o u n d pH =5, t o be i n v o l v e d w i t h t h e a d s o r p t i o n p r o c e s s e s . The enamel and h y d r o x y a p a t i t e e x t r a c t s showed b e t t e r I E F d e f i n i t i o n s a n d a r e c o n s i d e r e d t o a d s o r b p r o t e i n s b e t t e r . Some c o n c e r n c a n a l w a y s be r a i s e d r e g a r d i n g p o s s i b l e c a r r y o v e r f r o m supernatant used i n a d s o r p t i o n , through r i n s i n g , and i n t o extraction s o l u t i o n s . In adsorption experiments designed w i t h t h e a g a r o s e g e l s , more t h o r o u g h r i n s i n g s o f t h e powders were p e r f o r m e d p r i o r t o e x t r a c t i o n . The r i n s i n g s c o n s i s t e d o f t h r e e s e p a r a t e 5 ml H Q r i n s e s and w i t h e a c h v i g o r o u s l y s h a k e n i n t u b e w i t h m i x e r . The I E F p a t t e r n s w i t h t h e a g a r o s e p l a t e s w e r e n o t t h a t much d i f f e r e n t t h a n obtained w i t h p o l y a c r y l a m i d e . These r e s u l t s a r e i n l i n e w i t h a study (26) i n d i c a t i n g t h e IEF p a t t e r n s o f t h e e x t r a c t e d p r o t e i n s from nine h y d r o x y a p a t i t e s w i t h v a r i o u s s u r f a c e p r o p e r t i e s were a l l s i m i l a r and w i t h i n t h e a c i d i c pH r a n g e o b s e r v e d h e r e . The h i g h c a r b o h y d r a t e c o n t e n t s a n a l y z e d by FT-IR on a l l i n - v i v o exposed s u r f a c e s a r e l i k e l y due t o products of b a c t e r i a that c o l o n i z e t h e surfaces a f t e r s e v e r a l days 2

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27.

MUELLER

Characterization of the Acquired Biofilms

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o f i n - v i v o u s a g e . Hence, f o r t h e s e s u r f a c e s i n f o r m a t i o n o b t a i n e d i s l i k e l y t o be d i f f e r e n t t h a n f o r a l l o y s e x p o s e d f o r s h o r t t i m e s . The v a r i a t i o n s i n p r o t e i n t o c a r b o h y d r a t e r a t i o s f o r s u r f a c e s c r a p i n g s from d i f f e r e n t s i t e s on a l l o y s u r f a c e e x p o s e d t o s a l i v a f o r o n l y 1 hour a r e l i k e l y from a d s o r p t i o n o f d i f f e r e n t amounts o f t h e same s p e c i e s , o r a d s o r p t i o n of d i f f e r e n t types o f p r o t e i n s , carbohydrates, o r g l y c o p r o t e i n s . Hence, t h i s d a t a r e f l e c t s d i r e c t l y t h e nature of the adsorbing species. The i m p o r t a n c e o f t h e r e l e a s e d c o r r o d e d i o n s from a l l o y s i n a f f e c t i n g a d s o r p t i o n must be s t r e s s e d . I t was s h o w n . ( 2 7 ) t h a t f o r a v a r i e t y o f m e t a l l i c s a l t s added t o whole s a l i v a t h a t C u * and Z n g e n e r a t e d p r e c i p i t a t e s w i t h p r o t e i n s w h i c h were most s i m i l a r t o whole s a l i v a . The p r e c i p i t a t e s w i t h C a * were m i s s i n g some o f t h e p r o t e i n bands and t h e p r e c i p i t a t e s w i t h S r * . B a * , F e * , F e * , and Α Γ - c o n t a i n e d o n l y a s t r o n g a c i d i c p r o t e i n . Some c a t i o n s l i k e C u - and S n * a r e a l s o c o n s i d e r e d (3) t o i n h i b i t a d s o r p t i o n through t h e i r b i n d i n g t o charged groups on p e l l i c l e o r b a c t e r i a . Comparison o f t h e IEF p a t t e r n s o b t a i n e d i n t h i s study t o t h e r e s u l t s o b t a i n e d (25) w i t h human p a l a t i n e s a l i v a i n d i c a t e s g e n e r a l agreement. About t h e same number o f p r o t e i n bands o c c u r r e d w i t h b o t h s t u d i e s and w i t h a c i d i c p r o t e i n s b e i n g t h e most i n t e n s e i n t h e p r o t e i n p a t t e r n s . 2

2

2

3

2

2

3

3

2

2

Acknowledgments The FT-IR s p e c t r u m o f t h e r e t r i e v e d i n - v i v o s a m p l e s were courtesy o f t h e National Center f o r Biomedical I n f r a r e d Spectroscopy, B a t t e l l e L a b o r a t o r i e s , Columbus, O h i o . T h i s i n v e s t i g a t i o n was s u p p o r t e d i n p a r t by USPHS R e s e a r c h G r a n t DE 05761 from t h e N a t i o n a l I n s t i t u t e o f D e n t a l R e s e a r c h , B e t h e s d a M D , 20205.

Literature Cited 1. Ericson, T.; Pruitt, Κ. M.; Arwin, Η.; Lundstrom, I. Acta Odontol. Scand. 1982, 40, 197-201. 2. Baier, R. Ε.; Glantz, P. -O. Acta Odontol. Scand. 1978, 36, 289-301. 3. Skjorland, Κ. Acta Odontol. Scand. 1982, 40, 129-34. 4. Sonju, T.; Christensen, T. B.; Knrnstad, L . ; Rolla, G. Caries Res. 1974, 8, 113-22. 5. Bennick, Α.; Cannon, M.; Madapallimattam, G. Caries Res. 1981, 15, 9-20. 6. Vogel, J. C.; Belcourt, Α.; Gillmeyh, S. Caries Res. 1981, 15, 243-49. 7. Rolla, G. Swed. Dent. J. 1977, 1, 241-51. 8. Ingersoll, C. E. J. Dent. Res. 1976, 55, Pt121(B). 9. Tabak, L. A.; Levine, M. J.; Mandel. I. D.; Ellison, S. A. J. Oral Path. 1982, 11, 1-17. 10. Hay, D. I. Arch. Oral Biol. 1967, 12, 937-46. 11. Meckel, A. H. Arch Oral Biol. 1965, 10, 585-97. 12. Pruitt, Κ. M. Swed. Dent. J. 1977, 1, 225-40. 13. Tabak, L. A.; Levine, M. J., Jain, Ν. Κ.; Bryan, A. R.; Cohen, R. Ε.; Monte, L. D.; Zawacki, S.; Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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14. 15. 16. 17. Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 1, 2016 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch027

18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

PROTEINS AT INTERFACES

Nancollas, G. H.; Slomiany, Α.; Slomiany, B. L. Arch. Oral Biol. 1985, 30, 423-7. Mayhall, C. W. Arch. Oral Biol. 1970, 15, 1327-41. Hay, D. I. Arch. Oral Biol. 1973, 18, 1517-29. Leach. S. Α.; Lyon, R.; Appleton, J. In Surface and Colloid Phenomena in the Oral Cavity: Methodological Aspects; IRL Press Ltd.: London, 1982; pp 63-78. Hannesson Eggen, Κ : Rolla, G. Scand. J. Dent. Res. 1982, 90, 182-88. Bennick, Α.; Chau, G.: Goodlin, R.; Abrams, S.; Tustian, D.; Mandapallimattam, G. Arch. Oral Biol. 1983, 28, 19-27. Stiefel, D. J. J. Dent. Res. 1976, 55, 66-73. Juriaanse. A. C.; Booij. M.; Arends, J.; Ten Bosch, J. J. Arch. Oral Biol. 1981, 26, 91-6. Arends. J.; Jongebloed, W. L. Swed. Dent. J. 1977, 1, 215-24. Ellison, S. A. In Saliva and Dental Caries; Kleinberg, I.; Ellison, S. Α.; Mandel. I. D., Ed.; Sp. Supp. Microbiology Abstracts, 1979; pp 13-29. Sonju, T.; Glantz. P. -O. Arch. Oral Biol. 1975, 20, 687-91. Mueller, H. J.; Barrie, R. M. J. Dent. Res. 1985. 64. Pt 1753. Shiba, Α.; Sano, Κ.; Nakao, M.; Kobayashi, Κ.; Igarashi, Y. Arch. Oral Biol. 1983, 28, 363-4. Wilkes, P. D.; Leach, S. A. J. Dent. 1979, 7, 213-20. Voegel, J. C.; Belcourt, A. Arch. Oral Biol. 1980, 25, 137-9.

Received January 3, 1987

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.