Adhesives from Marine Mussels

Chapter 33 Adhesives from Marine Mussels Christine V. Benedict and P a u l T. Picciano BioPolymers, Inc. P.O. Box 1307 Farmington, C T 06034

The common blue mussel, Mytilus edulis L., has evolved an adhesive mechanism that performs optimally in the marine environment. The functional unit of this system is a polyphenolic protein (MAP) rich in 3,4-dihydroxyphenylalanine (L-dopa) and lysine. Several systems that demonstrate the utility of this adhesive protein have been developed. This extracted protein alone is a highly efficient mediator of the attachment of mammalian, yeast, and bacterial cells to inert substrates. Two-part adhesive formulations were examined in situ for tissue repair efficacy on ocular tissues. In vitro, corneal permeability studies showed that the ocular formulation of MAP freely allows the diffusion of even high molecular weight (42K) moieties, and that proteolytic enzymes will attack the adhesive matrix. The biocompatibility of MAP alone and MAP adhesive formulations was evaluated using mammalian cell growth rate and cell growth inhibition assays in vitro. The environment of the sea is similar in many ways to the internal environment of mammalian organisms. Tissues are bathed influidswith a pH and ionic and enzymatic composition similar to saltwater. Theoretically, the attachment mechanisms that some marine invertebrates have evolved for survival should be useful as surgical or wound repair adhesives in vivo. Best u n d e r s t o o d o f the invertebrate adhesive-mediated a t t a c h m e n t m e c h a n i s m s is t h a t o f the c o m m o n blue mussel, Mytilus edulis, a n d its close r e l a t i v e , Mytilus californianus L . T h e " m e c h a n i s m " includes t h e byssus, a n a c e l l u l a r p r o t e i n a ceous o r g a n p r o d u c e d b y glands inside the mussel, c o m b i n e d w i t h a d e l i v e r y syst e m t h a t secretes the byssus efficiently u n d e r w a t e r . T h e p r o t e i n t h a t is the funct i o n a l u n i t o f the adhesive m i x t u r e was first p u r i f i e d f r o m the g l a n d where i t o r i g inates a n d characterized b y W a i t e a n d T a n z e r ( i ) . C a l l e d mussel adhesive p r o t e i n ( M A P ) , i t is a h i g h m o l e c u l a r weight (120,000 ± 10,000 M W ) basic p r o t e i n , 0097-6156/89/0385-0465$06.00/0 © 1989 American Chemical Society

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rich i n lysine, hydroxylated (L-dopa).

amino

acids,

and

3,4-dihydroxyphenylalanine

W h i l e the c o m p o s i t i o n a n d sequence of the a m i n o acids have been k n o w n since 1983 (2,3), m e t h o d s for increased-scale e x t r a c t i o n were not developed u n t i l 1985. T h i s scaled p r o d u c t i o n has allowed for the development of s i n g l e - p a r t a d hesive systems ( C e l l - T a k adhesive) for the i m m o b i l i z a t i o n of b i o l o g i c a l l y active moieties t o inert substrates. It has also p e r m i t t e d research o n t w o - p a r t adhesive f o r m u l a t i o n s for the b o n d i n g of tissues. T h i s paper specifically addresses the b i o c o m p a t i b i l i t y issue w i t h descriptions o f the i m m o b i l i z a t i o n of cells t o C e l l T a k p r o t e i n - c o a t e d p l a s t i c w a r e , m e t h o d s for w o u n d closure, a n d p r e l i m i n a r y toxicology data. Experimental

Methodology

C e l l A t t a c h m e n t M u s s e l A d h e s i v e P r o t e i n ( M A P ) . M A P has been spe c i a l l y f o r m u l a t e d for delivery t o a n inert substrate i n single-component f o r m for the i m m o b i l i z a t i o n of b i o l o g i c a l l y active m a t e r i a l s ( C e l l - T a k ) . T h r e e m a m m a l i a n cell types were used t o c o m p a r e a t t a c h m e n t t o C e l l - T a k adhesive w i t h a t t a c h m e n t t o u n c o a t e d tissue c u l t u r e p l a s t i c w a r e a n d p l a s t i c w a r e c o a t e d w i t h other c o m m e r c i a l l y available a t t a c h m e n t factors. B a b y h a m s t e r k i d n e y cells ( B H K - 2 1 ; A T C C C C L 10) were g r o w n i n m o d i f i e d E a g l e ' s m e d i u m ( M E M ) c o n t a i n i n g 1 0 % c a l f s e r u m a n d 1 0 % t r y p t o s e phosphate b r o t h . H u m a n h i s t i o c y t i c l y m p h o m a cells ( U - 9 3 7 ; A T C C C R L 1593) were g r o w n i n R P M 1 1640 m e d i u m c o n t a i n i n g 1 0 % c a l f s e r u m . B o v i n e corneal e n d o t h e l i a l cells ( B C E ) d e r i v e d f r o m p r i m a r y isolates were m a i n t a i n e d i n M E M p l u s 1 5 % c a l f s e r u m . T h e a t t a c h ment of t w o b a c t e r i a , Staphylococcus aureus a n d Escherichia coli, a n d one yeast, Saccharomyces cerevisiae, to M A P - c o a t e d p l a s t i c w a r e a n d u n c o a t e d p l a s t i c w a r e was also e v a l u a t e d . A l l three organisms were g r o w n i n t r y p t i c a s e soy b r o t h . C e l l - T a k p r o t e i n was coated o n tissue c u l t u r e p l a s t i c w a r e b y a s o l u t i o n cast i n g m e t h o d . F o r a l l e x p e r i m e n t s , 5 μL of the 10 m g / m L s o l u t i o n was spread a n d d r i e d o n t o 3 5 - m m , 1 0 - c m p l a s t i c dishes for a final density of 5 / i g / c m . A f ter b e i n g a i r d r i e d , the plates received one e t h a n o l ( 9 5 % v / v ) a n d two d i s t i l l e d water rinses. A l l other a t t a c h m e n t factors were used a c c o r d i n g to m a n u f a c t u r ers' r e c o m m e n d a t i o n s . C o l l a g e n ( E t h i c o n , Inc., S o m e r v i l l e , N J ) - c o a t e d plates were p r e p a r e d b y d i l u t i n g one p a r t c o l d (4 ° C ) collagen d i s p e r s i o n (6 m g / m L ) i n t o s i x p a r t s of c o l d 5 0 % m e t h a n o l . T h i s m i x t u r e was v o r t e x e d v i g o r o u s l y for several m i n u t e s a n d p i p e t t e d o n t o a tissue c u l t u r e d i s h so t h a t o n l y the b o t t o m of the d i s h was covered. W i t h i n 20 seconds, the collagen was removed by a s p i r a t i o n , a n d the d i s h was inverted s u c h t h a t i t rested against a l i d at a 30° angle. F o l l o w i n g 1 h r of u n d i s t u r b e d d r y i n g i n a l a m i n a r flow h o o d , the dishes were ready for use. L a m i n i n ( C o l l a b o r a t i v e R e s e a r c h , L e x i n g t o n , M A ) was s u p p l i e d i n 1-mg q u a n t i t i e s i n 1 m L of 50 m M T r i s s o l u t i o n i n p h y s i o l o g i c a l saline. F o l l o w i n g a slow t h a w o f l a m i n i n s o l u t i o n at 0 t o 4 ° C f r o m -20 ° C storage, 10 t o 15 / i g of l a m i n i n s o l u t i o n was p i p e t t e d o n t o tissue c u l t u r e p e t r i 2

2

33.

B E N E D I C T A N D

PicciANO

Adhesives from Marine

Mussels

467

dishes i n 0.5 m L of 0 . 0 1 M phosphate buffer, p H 7.4, the dishes were d r i e d at 37 ° C . I m m e d i a t e l y u p o n d r y i n g , the dishes were prepared for use. F i b r o n e c t i n ( C o l l a b o r a t i v e R e s e a r c h , L e x i n g t o n , M A ) was s u p p l i e d i n 1-mg q u a n t i t i e s as a l y o p h i l i z e d powder. P r i o r t o use, f i b r o n e c t i n was allowed t o e q u i l i b r a t e to r o o m t e m p e r a t u r e after 4 ° C storage. T h e powder was r e c o n s t i t u t e d w i t h 1 - m L ster ile d i s t i l l e d water a n d allowed t o s t a n d for 30 m i n for s o l u b i l i z a t i o n . T w e n t y μg of fibronectin were a d d e d t o each tissue c u l t u r e d i s h i n 0.5 m L a n d allowed to a i r dry. A t t h i s t i m e , the d i s h was ready for cell seeding. H i g h - m o l e c u l a r weight p o l y - D - l y s i n e ( C o l l a b o r a t i v e R e s e a r c h , L e x i n g t o n , M A ) was s u p p l i e d i n q u a n t i t i e s of 5-mg l y o p h i l i z e d p o w d e r . P r i o r t o use, t h i s p o w d e r was allowed t o e q u i l i b r a t e t o r o o m t e m p e r a t u r e after 4 ° C storage before s o l u b i l i z a t i o n . Dishes were coated w i t h 50 μg i n 1 m L of sterile d i s t i l l e d water a n d allowed t o s t a n d at r o o m t e m p e r a t u r e for 5 m i n . A t t h a t t i m e , the s o l u t i o n was a s p i r a t e d a n d the dishes r i n s e d two t i m e s w i t h 1.5 m L of sterile d i s t i l l e d w a t e r . F o l l o w i n g each rinse, l i q u i d was a s p i r a t e d for complete r e m o v a l of u n a t t a c h e d p o l y m e r . T h e dishes were d r i e d a n d used i m m e d i a t e l y . T h e a t t a c h m e n t assays were designed t o q u a n t i f y a t t a c h e d cells after v a r i o u s i n c u b a t i o n p e r i o d s at 37 ° C . B H K a n d B C E cells were t r y p s i n i z e d f r o m stock plates, washed i n fresh m e d i u m b y c e n t r i f u g a t i o n , a n d suspended i n fresh R P M 1 1640 w i t h 1 0 % c a l f s e r u m at a density of 2 χ 1 0 c e l l s / m L . U - 9 3 7 cells, w h i c h are g r o w n i n s u s p e n s i o n , were washed b y c e n t r i f u g a t i o n a n d resuspended t o s i m i l a r densities. Suspensions were seeded o n t o u n t r e a t e d tissue c u l t u r e dishes (control) a n d dishes t r e a t e d w i t h a t t a c h m e n t factors. A t 5, 12.5, a n d 20 m i n , t r i p l i c a t e e x p e r i m e n t a l a n d c o n t r o l plates were chosen at r a n d o m for q u a n t i f i c a t i o n of u n a t t a c h e d cells. T h e s e were removed f r o m the dishes after gentle a g i t a t i o n and counted on a hemacytometer. D a t a were c a l c u l a t e d as percent of cells a t t a c h e d b y s u b t r a c t i n g the n u m b e r of u n a t t a c h e d cells o b t a i n e d f r o m dishes (average of three) f r o m the t o t a l n u m b e r of cells p l a t e d , d i v i d i n g the result b y the t o t a l n u m b e r of cells p l a t e d , a n d m u l t i p l y i n g the q u o t i e n t by 1 0 0 % . A s i m i l a r t y p e of assay was done w i t h b a c t e r i a l a n d yeast cultures t r e a t e d s i m i l a r l y . C u l t u r e s were washed b y c e n t r i f u g a t i o n i n 0.1 M p h o s p h a t e buffer ( p H = 7.0) a n d resuspended t o a n O D 8 o = 0.3. A t t a c h m e n t after 30 m i n at r o o m t e m p e r a t u r e to p l a s t i c a n d C e l l - T a k - c o a t e d p l a s t i c was e v a l u a t e d q u a l i t a t i v e l y by m i c r o s c o p i c v i s u a l i z a t i o n . T h e g r o w t h rate of m a m m a l i a n cells i n the presence of C e l l - T a k adhesive was assayed t o evaluate any p o t e n t i a l adverse effects caused b y t h i s p r o t e i n . B o v i n e corneal e n d o t h e l i a l cell ( B C E ) stocks were g r o w n t o confluency i n M E M p l u s 1 5 % c a l f s e r u m , t r y p s i n i z e d , a n d washed several t i m e s b y c e n t r i f u g a t i o n o n M E M . Suspensions (5 χ 1 0 c e l l s / m L ) were seeded o n t o u n t r e a t e d 3 5 - m m dishes (control) a n d dishes w i t h C e l l - T a k p r o t e i n (5 / i g / c m ) i n M E M w i t h 1 5 % c a l f s e r u m . A t various t i m e p o i n t s d u r i n g the i n c u b a t i o n at 37 ° C w i t h 5 % CO2, t r i p l i c a t e plates were removed. T h e a t t a c h e d cells were t h e n t r y p s i n i z e d f r o m the surface, washed, a n d counted i n a h e m a c y t o m e t e r . 5

5

4

2

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ADHESIVES F R O M R E N E W A B L E RESOURCES

O c u l a r T i s s u e B o n d i n g In Vitro. T w o m o d e l systems were designed for the e v a l u a t i o n of M A P adhesives in vitro. B o v i n e eyes o b t a i n e d the same d a y f r o m a b a t t o i r s were always used. T h e first s y s t e m was developed t o d e m o n s t r a t e the a b i l i t y of M A P t o s t r o n g l y adhere t o c o r n e a l s t r o m a as a prelude t o r e p a i r i n g w o u n d s in situ. This s y s t e m also assisted i n the development of M A P f o r m u l a t i o n s a n d p r o t o c o l s for use. E n d o t h e l i a l a n d e p i t h e l i a l cells were removed f r o m freshly i s o l a t e d b o v i n e cornea b y s c r a p i n g , a n d a 1 - c m area f r o m each of two sections of cornea was t r e a t e d w i t h 50 μg of M A P . T h e sections were b o n d e d b y o v e r l a p p i n g the two s t r i p s a n d a l l o w i n g t h e m t o set (or cure) at r o o m t e m p e r a t u r e . T h e tissue was kept m o i s t a n d flat b y o v e r l a y i n g w i t h a d i s t i l l e d w a t e r - f i l l e d d i a l y s i s b a g . A f t e r 20 m i n , the corneal b o n d was subjected t o a shear test t o determine adhesive s t r e n g t h . O n e e n d of the overlap j o i n t was suspended v e r t i c a l l y f r o m a c l a m p , a n d a second c l a m p was a t t a c h e d t o the lower e n d together w i t h a n e m p t y p l a s t i c b a g . W e i g h t was increased t o the j o i n t at a constant rate of 14 g / m i n b y the a d d i t i o n of water t o the b a g u s i n g a p e r i s t a l t i c p u m p . A t j o i n t f a i l u r e , the water flow was t u r n e d off, a n d the b a g was weighed t o y i e l d b o n d s t r e n g t h . A d h e s i v e s t r e n g t h was considered t o be the m a x i m u m weight of water suspended d i v i d e d by the b o n d area a n d is represented i n g / c m . T h e second m o d e l s y s t e m was designed t o d e m o n s t r a t e the f e a s i b i l i t y of u s i n g M A P adhesives i n sealing s m a l l a n d large tissue p e r f o r a t i o n s . E p i t h e l i a l cells f r o m w h o l e b o v i n e eyes were removed w i t h a s c a l p e l f r o m a 15- t o 20m m region of the cornea. A p e r f o r a t i o n was prepared b y l a c e r a t i n g the center of the s c r a p e d cornea w i t h a s c a l p e l . A n 18-gauge needle a t t a c h e d t o a 10m L syringe c o n t a i n i n g saline was inserted i n t o the a n t e r i o r c h a m b e r at the c o r n e a l / s c l e r a l j u n c t i o n . A f u l l thickness p e r f o r a t i o n was assured b y i n s e r t i n g saline i n t o the anterior chamber a n d l o o k i n g for fluid leakage t h r o u g h the corneal p u n c t u r e . T h e s c r a p e d area was t h e n r i n s e d w i t h deionized water a n d excess water is removed b y s w a b b i n g . M A P adhesive (50 / i g / c m ) was t h e n a p p l i e d i m m e d i a t e l y p e r i p h e r a l t o the p e r f o r a t i o n site. A h y d r o g e l t h e r a p e u t i c contact lens ( H y p a n , K i n g s t o n Technologies, Inc.) t h a t h a d been presoaked for 30 m i n i n phosphate-buffered saline ( P B S ) was o v e r l a i d onto the w o u n d site a n d gentle pressure a d d e d t o the lens-corneal interface t o ensure direct a p p o s i t i o n of the p a t c h t o the tissue. A dialysis* b a g was a p p l i e d over the j o i n t for 20 m i n d u r i n g c u r i n g . S t r e n g t h o f b o n d was measured u s i n g a m a n o m e t e r a t t a c h e d t o the needle inserted i n t o the anterior c h a m b e r . T h e d i a l y s i s b a g was removed a n d the eye pressurized at a b o u t 120 i n / m i n w i t h a s y r i n g e connected to the m a n o m e t e r , w h i l e leakage a n d pressure were m o n i t o r e d . T h e water pressure recorded was the r e a d i n g a t t a i n e d at the first sign o f leakage. T h e pressure was converted t o m m H g b y d i v i d i n g b y 0.535. D a t a represent the average o f at least two assays. 2

2

B i o c o m p a t i b i l i t y a t t h e C e l l C u l t u r e L e v e l . T h e e v a l u a t i o n of c y t o t o x i c i t y o n a cell c u l t u r e agar overlay f o l l o w i n g the a p p l i c a t i o n of l i q u i d M A P , l i q u i d e n z y m a t i c crosslink c a t a l y s t , a n d e x t r a c t s o f crosslinked solids was done t o de-

33.



PicciANO

Mussels

469

tect the response of a m a m m a l i a n cell c u l t u r e monolayer (mouse L - 9 2 9 , A T C C C C L 81) t o r e a d i l y diffusible components of the p r e p a r a t i o n s . L i q u i d M A P (4.0 m g / m L ) i n water a n d catechol oxidase (4.5 m g / m L , S i g m a C h e m i c a l C o m p a n y ) i n 0 . 1 M p h o s p h a t e buffer were used as i s . T w o f o r m u l a t i o n s , one h i g h crosslinked (15 U e n z y m e : l μg M A P ) a n d one low crosslinked (5 U e n z y m e : l μg M A P ) , were p r e p a r e d b y a l l o w i n g the r e a c t i o n t o occur for 1 h r at r o o m t e m p e r a t u r e followed b y quick freezing a n d l y o p h i l i z a t i o n . T h e r e s u l t a n t flakes were e x t r a c t e d w i t h i r r i g a t i o n saline ( U S P ) , cottonseed o i l , a n d e t h a n o l ( 9 5 % v / v ) at 2 0 - m L solvent per 6.0-mg flakes at 60 ° C for 4.5 h r . T h e s u p e r n a t a n t s were used i n the assay w i t h i n 24 h r . T h e mouse L - 9 2 9 fibroblast l i n e was c u l t i v a t e d i n E a g l e ' s M i n i m a l E s s e n t i a l M e d i u m ( M E M ) p l u s 1 0 % c a l f s e r u m . C e l l s were seeded i n 1 0 0 - m m - d i a m e t e r cell c u l t u r e plates at 4 χ 1 0 cells per p l a t e a n d allowed t o b e c o m e established for 24 h r p r i o r t o use. A f t e r the monolayer was w a s h e d , 10 m L of a n agar overlay c o n s i s t i n g of 2 % B a c t o - A g a r a n d 2 χ M E M was a d d e d t o each p l a t e a n d allowed t o solidify. 6

T h e e x t r a c t s a n d l i q u i d components were delivered i n d u p l i c a t e t o the agar as 2 0 - / i L l i q u i d o n 1-cm-diameter W h a t m a n N o . 2 filter p a p e r . T h e solids (100 /ig) were i m p l a n t e d d i r e c t l y o n t o the agar. N e g a t i v e controls i n c l u d e d filter paper w i t h 20 / i L of each solvent a n d filter paper w i t h no solvent. T h e p o s i t i v e c o n t r o l was p o l y v i n y l c h l o r i d e ( P V C ) sheeting c o n t a i n i n g 1% t i n s t a b i l i z e r ( T r a v e n o l L a b o r a t o r i e s , I n c . ) , cut i n t o 1-cm-diameter disks a n d i m p l a n t e d d i r e c t l y o n t o the agar. P l a t e s were i n c u b a t e d at 37 ° C i n a h u m i d i f i e d i n c u b a t o r w i t h 5 % CO2 for 24 h r . A t t h i s t i m e , a l l plates were fixed w i t h 1 0 % buffered f o r m a l i n a n d r i n s e d w i t h w a t e r , the agar was peeled off, a n d the cells were s t a i n e d w i t h 0 . 1 % n e u t r a l r e d s o l u t i o n for 15 m i n . T h e areas of l y s i s zones were m e a s u r e d , a n d lysis indices were c a l c u l a t e d as described l a t e r i n t h i s p a p e r (see T a b l e H I i n the results section). A p p e a r a n c e of the monolayer s u r r o u n d i n g the test or c o n t r o l s a m p l e was described as n o n t o x i c (same as u n t r e a t e d m o n o l a y e r ) , s l i g h t l y t o x i c ( p i n k b u t not as d a r k l y s t a i n e d as u n t r e a t e d m o n o l a y e r ) , or t o x i c (very faint p i n k or n o color). A q u a n t i t a t i v e a p p r o a c h was t a k e n to evaluate the effect of v a r i o u s concen t r a t i o n s of water e x t r a c t s of the two crosslinked solids a n d i n d i v i d u a l l i q u i d c o m p o n e n t s o n cell g r o w t h of L - 9 2 9 cells in vitro. B o t h cells a n d adhesive m a terials were prepared as described above. F o r t h i s assay, d i l u t i o n s of a n aqueous e x t r a c t of the solids were p r e p a r e d for a dose-response e v a l u a t i o n . T h e f o l l o w i n g weight per v o l u m e r a t i o s were used: 4,000, 500, 100, 50, 4, 3, 2, 1 μg per 2 0 - m L water. E x t r a c t i o n was p e r f o r m e d for 4 h r at 60 ° C followed b y 20 h r at r o o m t e m p e r a t u r e . S u p e r n a t a n t s were transferred t o clean containers. T h e negative c o n t r o l consisted of sterile, t r i p l e d i s t i l l e d water, a n d the p o s i t i v e c o n t r o l was 40 m g / m L d e x t r a n sulfate. M o u s e fibroblast cells ( L - 9 2 9 ) , t r y p s i n i z e d f r o m stock plates, were suspended i n complete E a g l e ' s M e d i u m at a density of 1 0 cells per m L . F o r the assay, l i q u i d e x t r a c t s ( 1 - m L final v o l u m e ) were m i x e d w i t h 1 - m L d o u b l e s t r e n g t h M E M , t o 6

470

ADHESIVES F R O M RENEWABLE R E S O U R C E S

w h i c h 0.2 m L of the cell suspension (2 χ 1 0 cells) was a d d e d at t i m e zero. T e n replicates were m a d e for each e x t r a c t . I m m e d i a t e l y after m i x i n g , five t u b e s were centrifuged a n d the cells washed a n d resusp ended i n p h o s p h a t e buffered saline ( P B S ) a n d stored at 4 ° C for zero t i m e reference p o i n t s . A f t e r 72-hr i n c u b a t i o n at 37 ° C i n a h u m i d i f i e d i n c u b a t o r , d u r i n g w h i c h t i m e the cells h a d g r o w n i n monolayers, the r e m a i n i n g tubes were decanted, the monolayers were washed g e n t l y a n d t r y p s i n i z e d . T h e cells were recovered a n d washed t w i c e b y centrifugation w i t h P B S . T h e extent of cell g r o w t h as i n d i c a t e d b y t o t a l p r o t e i n c o n c e n t r a t i o n was done u s i n g the F o l i n - P h e n o l m e t h o d of L o w r y (4) w i t h a b o v i n e s e r u m a l b u m i n s t a n d a r d o n cell samples s o l u b i l i z e d w i t h 0 . 0 5 % s o d i u m l a u r y l sulfate. D a t a are presented as percent i n h i b i t i o n o f cell g r o w t h relative to u n t r e a t e d c o n t r o l s . 5

Results C e l l A t t a c h m e n t t o M A P . T h e a t t a c h m e n t of t w o c e l l types, one anchoragedependent a n d the other anchorage-independent, was c o m p a r e d over t i m e o n C e l l - T a k adhesive-coated dishes w i t h the a t t a c h m e n t o n other c o m m e r c i a l l y available factors. B H K - 2 1 cells n o r m a l l y a t t a c h a n d grow i n monolayers. F i g ure 1 A g r a p h i c a l l y shows the a t t a c h m e n t of B H K - 2 1 cells, w h i c h a t t a i n e d 70 t o 9 0 % efficiency i n 20 m i n t o l a m i n i n , fibronectin, a n d C e l l - T a k adhesive a n d 6 0 % t o p o l y - D - l y s i n e . M a x i m u m a t t a c h m e n t o c c u r r e d at a faster rate o n C e l l T a k p r o t e i n , where 9 0 % efficiency was achieved i n 12.5 m i n . T h e anchoragei n d e p e n d e n t , or suspension cell l i n e , U - 9 3 7 , achieved 7 5 - 8 5 % a t t a c h m e n t effi ciency o n C e l l - T a k adhesive ( F i g u r e I B ) b u t less t h a n 3 0 % a t t a c h m e n t to any o f the other factors. T h e m e c h a n i s m b y w h i c h C e l l - T a k p r o t e i n accomplishes t h i s efficiency at the m o l e c u l a r level is addressed w i t h the o b s e r v a t i o n t h a t different cell types a t t a c h w i t h the same k i n e t i c s ( F i g u r e 2 A ) . T h e l y m p h o m a l i n e ( U - 9 3 7 ) , B H K cells, a n d b o v i n e c o r n e a l e n d o t h e l i a l cells a t t a c h w i t h v a r y i n g efficiencies t o p l a s t i c ( F i g u r e 2 B ) b u t e x h i b i t the same rate a n d o v e r a l l efficiency o n C e l l - T a k p r o t e i n . T h e a t t a c h m e n t lacks the specificity t h a t w o u l d i n d i c a t e r e c e p t o r - m e d i a t e d a t t a c h ment a n d s u p p o r t s the r o l l o f nonspecific i n t e r a c t i o n s . M o r e o v e r , other d a t a (not s h o w n ) suggest t h a t the a t t a c h m e n t o f cells is due t o C e l l - T a k a n d not t o a n y c o m p o n e n t such as fibronectin t h a t is f o u n d i n s e r u m . T h i s is seen i n e x p e r i m e n t s i n v o l v i n g the p r e i n c u b a t i o n o f C e l l - T a k - c o a t e d dishes w i t h either fibronectin or s e r u m . C e l l - T a k - c o a t e d plates were p r e i n c u b a t e d w i t h c e l l c u l t u r e m e d i u m c o n t a i n i n g 2 0 % c a l f s e r u m for 30 m i n , a n d fibronectin was d r i e d onto other plates coated w i t h C e l l - T a k before U - 9 3 7 cells were p l a t e d . C e l l attachment on fibronectin-coated C e l l - T a k plates was i d e n t i c a l to a t t a c h m e n t t o C e l l - T a k alone, a n d a t t a c h m e n t efficiency was i n h i b i t e d b y 4 0 % o n plates preincubated with serum. T h e i m m o b i l i z a t i o n o f m i c r o o r g a n i s m s t o C e l l - T a k p r o t e i n was m o n i t o r e d m i c r o s c o p i c a l l y . F i g u r e s 3, 4, a n d 5 show the sparse a t t a c h m e n t t o p l a s t i c

33.


0

PicciANO

4


8

Mussels

12

16

471

20

Time (minutes)

90

Time (minutes)

F i g u r e 1. T h e a t t a c h m e n t of adherent B H K - 2 1 cells ( F i g u r e 1 A ) herent U - 9 3 7 cells ( F i g u r e I B ) to C e l l - T a k adhesive, • ; collagen, l y s i n e , + ; l a m i n i n , X ; fibronectin, Δ ; a n d p l a s t i c , V> monitored u n a t t a c h e d cells at 5, 12.5, a n d 20 m i n . D a t a were t h e n converted c a l l y t o percent a t t a c h e d of those seeded. w

a

s

and nonad Ο ; poly-Dby counting mathemati

472

ADHESIVES F R O M R E N E W A B L ER E S O U R C E S

100

Time (minutée)

F i g u r e 2. T h e a t t a c h m e n t k i n e t i c s of adherent B C E cells, Ο ; B H K - 2 1 cells, + ; a n d nonadherent U - 9 3 7 cells, • , were c o m p a r e d d i r e c t l y o n C e l l - T a k adhesive ( F i g u r e 2 A ) a n d u n c o a t e d tissue c u l t u r e p l a s t i c w a r e ( F i g u r e 2 B ) .

33.


PicciANO


Mussels

473

F i g u r e 3. T h e a t t a c h m e n t of E. coli t o p l a s t i c ( F i g u r e 3 A ) a n d C e l l - T a k adhesive ( F i g u r e 3 B ) was m i c r o s c o p i c a l l y c o m p a r e d at 3300 χ m a g n i f i c a t i o n .

F i g u r e 4. T h e a t t a c h m e n t of 5. aureus to p l a s t i c ( F i g u r e 4 A ) a n d C e l l - T a k adhesive ( F i g u r e 4 B ) was m i c r o s c o p i c a l l y c o m p a r e d at 3300 χ m a g n i f i c a t i o n .

F i g u r e 5. T h e a t t a c h m e n t of S. cerevisiae to p l a s t i c ( F i g u r e 5 A ) a n d C e l l - T a k adhesive ( F i g u r e 5 B ) was m i c r o s c o p i c a l l y c o m p a r e d at 3300 χ m a g n i f i c a t i o n .

474

ADHESIVES F R O M RENEWABLE RESOURCES

( A series) c o m p a r e d t o the enhanced a t t a c h m e n t w i t h C e l l - T a k p r o t e i n - c o a t e d p l a s t i c ( B series). T h e g r o w t h rate of B C E cells is not altered i n the presence of C e l l - T a k p r o t e i n ( F i g u r e 6). T h e same d a t a have been o b t a i n e d w i t h other adherent types ( B H K - 2 1 , d a t a not s h o w n ) . A f t e r a t t a c h m e n t , n o n a d h e r e n t cells do not grow as discrete colonies, p r e c l u d i n g the same t y p e o f e v a l u a t i o n w i t h U - 9 3 7 cells. O c u l a r T i s s u e B o n d i n g In Vitro. T h r e e factors were f o u n d t o be c r i t i c a l to the development of the m o d e l s y s t e m for w o u n d r e p a i r . T h e s e were the age of the eyes f o l l o w i n g e n u c l e a t i o n , the degree of c u r v a t u r e o f the cornea at the t i m e o f t e s t i n g , a n d the a m o u n t of adhesive p r o t e i n or r a t i o o f crosslinker t o p r o t e i n used t o close the w o u n d . E y e s used m o r e t h a n 20-hr postslaughter never y i e l d e d b o n d strengths i n excess of 30 g / c m . I n u n t r i m m e d corneas f r o m w h i c h b o t h the e p i t h e l i a l a n d e n d o t h e l i a l cell layers h a d been removed, a general t r e n d w i t h i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n was seen ( T a b l e I). W i t h higher a m o u n t s of 2

T a b l e I. C o r n e a t o C o r n e a B o n d S t r e n g t h s w i t h M A P Model Variable

MAP

Catechol Oxidase

Cornea scraped, not t r i m m e d

(μ&) 50 50 50

(U/M5) 13 16 18

50 50 56 58 75 75 100

16 18 16 10.5 13 18 18

106 72 86 82,100 46 8 3 , 79 105

Cornea trimmed, and scraped

52 52

9 12

72 183, 122, 2 6 1 , 89, 95

Cornea trimmed, not scraped

52

12

40

Breaking Load (g/cm ) 2

60 63 46

p r o t e i n , higher e n z y m e : s u b s t r a t e r a t i o s were r e q u i r e d t o achieve s i m i l a r b o n d strengths. T h e cornea has a n a t u r a l c u r v a t u r e ; w i t h the b o n d f o r m a t i o n a n d i n c u b a t i o n o n a flat surface, i t was f o u n d t h a t more r e p r o d u c i b l e d a t a were o b t a i n e d o n flatter, s c r a p e d , a n d t r i m m e d corneas. W i t h b e t t e r d e f i n i t i o n of

33.


PicciANO


uaawnNTraodo ocn

Mussels

475

476

ADHESIVES F R O M RENEWABLE R E S O U R C E S

the m o d e l s y s t e m , less p r o t e i n w i t h less crosslink c a t a l y s t achieved h i g h b o n d strengths. T h e a m o u n t of adhesive used was insufficient t o f o r m a n o p a q u e film between the t w o tissue pieces; the b o n d was clear, a n d c l a r i t y of the corneas appeared unchanged. T h e second m o d e l s y s t e m for M A P adhesion was designed to evaluate efficacy i n s e a l i n g s m a l l or large tissue p e r f o r a t i o n s i n a c l i n i c a l l y relevant m a n n e r . F u l l thickness corneal holes were created i n b o v i n e eyes in situ w i t h a s c a l p e l . I n t e r n a l fluid pressure caused aqueous h u m o r t o i m m e d i a t e l y flow out o f the eye w i t h consequent flattening of the cornea. A t r a n s p a r e n t h y d r o g e l disc ( H y p a n ) designed for use as a contact lens i n c o n j u n c t i o n w i t h M A P was used t o seal the p e r f o r a t i o n s . W i t h the corneas flat, s m a l l volumes (10-12 ^ L ) of M A P (5-6 m g / m L i n water) p l u s catechol oxidase (8-9 U / / x g M A P p r o t e i n ) were a p p l i e d a r o u n d the p e r i m e t e r o f the p e r f o r a t i o n f r o m w h i c h the e p i t h e l i a l cell layer h a d been r e m o v e d . T h e H y p a n disc ( 1 3 - m m d i a m e t e r , 0 . 2 - m m thickness) was t h e n centered over the p e r f o r a t i o n a n d gently s m o o t h e d out t o p e r m i t g o o d contact w i t h the cornea. A w a t e r - f i l l e d d i a l y s i s b a g was t h e n p l a c e d over the cornea to prevent desiccation d u r i n g the r o o m t e m p e r a t u r e i n c u b a t i o n . V a r i o u s cure t i m e s were tested, after w h i c h pressure was i n d u c e d b y water v i a a s y r i n g e connected t o a m a n o m e t e r , a n d b u r s t s t r e n g t h was recorded i n m m H g . T h e m e a s u r e m e n t s y s t e m h a d a n u p p e r l i m i t of a p p r o x i m a t e l y 110-120 m m H g ; i n the cases where the b o n d d i d not f a i l , the d a t a were recorded as m m H g ( T a b l e I I ) . B o n d cure was r a p i d w i t h m a x i m u m m e a s u r a b l e b o n d strengths achieved i n 10 m i n w i t h o n l y s l i g h t l y lower b o n d strengths seen at 5 m i n . A s w i t h the cornea overlap b o n d s , M A P at t h i s c o n c e n t r a t i o n p l u s the t r a n s p a r e n t H y p a n p r o d u c e d a seal t h a t was also clear.

T a b l e I I . C o r n e a l P e r f o r a t i o n Seal w i t h M A P Plus H y p a n Incubation T i m e (minutes)

Burst Strength ( m m Hg)

5

84, > 82

10

> 101, > 110, > 99

20

> 110, 64, > 112, > 116 116, 110

B i o c o m p a t i b i l i t y a t t h e C e l l C u l t u r e L e v e l . T h e l i q u i d components, M A P a n d catechol oxidase, a n d extracts o f two s o l i d crosslinked m a t e r i a l s were tested for cell c u l t u r e t o x i c i t y a n d g r o w t h i n h i b i t i o n . T h e e v a l u a t i o n of cell c u l t u r e c y t o t o x i c i t y i n a n agar overlay m e t h o d detects the i m p a c t o n cells o f any freely

33.


PicciANO


477

Mussels

diffusible c o m p o n e n t s or e x t r a c t able m a t e r i a l s . M o u s e fibroblasts ( L 9 2 9 ) were g r o w n i n monolayers, o v e r l a i d w i t h agar, a n d i n c u b a t e d for 24 h r w i t h filter paper discs i m p r e g n a t e d w i t h l i q u i d s o n the surface of the agar. S o l i d crosslinked m a t e r i a l s were p l a c e d d i r e c t l y o n the agar. T a b l e I I I shows the c y t o t o x i c i t y results o b t a i n e d i n t h i s s t u d y . P l a t e s re c e i v i n g n o t r e a t m e n t s t a i n e d a n even p i n k color, i n d i c a t i n g the presence of a h e a l t h y confluent cell monolayer. T h e p o s i t i v e c o n t r o l ( P V C ) p l a c e d i n each of the plates a l o n g w i t h test samples caused the expected t o x i c response w i t h greater t h a n 8 0 % l y s i s of the cells w i t h i n the zone s u r r o u n d i n g the samples. N e g a t i v e controls, w h i c h consisted of W h a t m a n N o . 2 q u a l i t a t i v e filter p a p e r (for s o l i d a n d l i q u i d samples) a n d the same filter paper i m p r e g n a t e d w i t h the a p p r o p r i a t e e x t r a c t i n g solvent (for e x t r a c t s ) , v a r i a b l y y i e l d e d s l i g h t l y p o s i t i v e results. S l i g h t t o x i c i t y i n the f o r m of a lighter s t a i n e d m o n o l a y e r was observed under three d r y filter papers (out of eight) a n d o n four filter papers i m p r e g n a t e d w i t h s a l i n e . T o x i c i t y was also evident s u r r o u n d i n g one of four filter papers i m p r e g n a t e d w i t h e t h a n o l . N o t o x i c i t y was observed under the four filter papers i m p r e g n a t e d w i t h cottonseed o i l . Table III. A g a r Overlay Cytotoxicity Results Test S u b s t a n c e

Positive Control

Test

Liquid M A P L i q u i d enzyme

T,T T,T

ST,ST,ST,ST Τ,Τ,Τ,Τ

ST,NT ST.NT

T,T T,T T,T T,T

ΝΤ,ΝΤ,ΝΤ,ΝΤ T,ST,ST,NT ST,ST,ST,NT ST,ST,ST,NT

ST,NT ST.NT ΝΤ,ΝΤ Τ,ΝΤ

T,T T,T T,T T,T

ΝΤ,ΝΤ,ΝΤ ST,ST,ST,NT ST,NT,NT,NT Τ,ΝΤ,ΝΤ,ΝΤ

ΝΤ,ΝΤ ΝΤ,ΝΤ

H i g h crosslinked: Solid Saline extract Cottonseed o i l extract E t h a n o l extract L o w crosslinked: Solid Saline extract Cottonseed oil extract E t h a n o l extract

1

Negative C o n t r o l

ΝΤ,ΝΤ ΝΤ,ΝΤ

*Τ = t o x i c ; N T = n o n t o x i c ; S T = s l i g h t l y t o x i c . O n e e n t r y for each s a m p l e .

F o u r samples were tested w i t h o u t e x t r a c t i o n or any other t r e a t m e n t : l i q u i d M A P , l i q u i d e n z y m e , h i g h crosslinked M A P s o l i d , a n d low crosslinked M A P s o l i d . T h e cell m o n o l a y e r under the l i q u i d M A P was s l i g h t l y l i g h t e r i n color t h a n the s u r r o u n d i n g monolayer (see "slight t o x i c i t y " i n T a b l e I I I ) . Since a s i m i l a r o b s e r v a t i o n was n o t e d for one of the negative controls, the l i q u i d M A P c a n be

478


considered t o be n o n t o x i c i n t h i s s y s t e m . W h e n s t a i n e d zones were e x a m i n e d u n d e r a n i n v e r t e d microscope, the few r e m a i n i n g adherent cells w i t h i n the zone a p p e a r e d t o be s w o l l e n t h o u g h not fragmented. T h e t o x i c i t y zone is c o m p a r a b l e t o t h a t o b t a i n e d w i t h the P V C p o s i t i v e c o n t r o l . N e i t h e r s o l i d m a t e r i a l s , h i g h a n d low crosslinked M A P , i n d u c e d any effect o n the cell monolayers. S a l i n e , cottonseed o i l , a n d e t h a n o l e x t r a c t s of the h i g h a n d low crosslinked s o l i d M A P showed low r e a c t i v i t y . A second i n d e x of b i o c o m p a t i b i l i t y was the q u a n t i t a t i v e a n a l y s i s of cell g r o w t h i n h i b i t i o n , a g a i n o n mouse fibroblast L 9 2 9 cells, i n d u c e d b y the l i q u i d c o m p o n e n t s of the adhesive s y s t e m a n d water extracts of t w o s o l i d crosslinked m a t e r i a l s . T a b l e I V is a s u m m a r y of the percent i n h i b i t i o n of cell g r o w t h (per cent I C G ) . T h e m e a n p r o t e i n values at 4 ° C have been s u b t r a c t e d f r o m the m e a n p r o t e i n values (five test samples) at 37 ° C for each t r e a t m e n t c o n d i t i o n . T h e percent i n h i b i t i o n of cell g r o w t h (percent I C G ) is s h o w n for each t r e a t m e n t c o n d i t i o n . T h e p r e c i s i o n of the assay is a p p r o x i m a t e l y ± 1 0 % . R e s u l t s for the negative a n d p o s i t i v e controls are s h o w n at the b o t t o m of the t a b l e . T u b e s r e c e i v i n g n o t r e a t m e n t , b u t h e l d at 4 ° C , h a d a m e a n p r o t e i n content of 89.7 |ig; after 72 h r of i n c u b a t i o n , the p r o t e i n content i n u n t r e a t e d tubes h a d increased t o 313.5 ^ g . T h e difference i n these t w o values (223.8 ^g) is t a k e n t o represent 1 0 0 % g r o w t h for t h i s e x p e r i m e n t . A p o s i t i v e c o n t r o l , D e x t r a n S u l f a t e , was tested at 40 m g / m L . A percent I C G greater t h a n 100 c a n result f r o m the c a l c u l a t i o n of t h i s value i f the p r o t e i n content of tubes h e l d at 4 ° C exceeds t h a t of the i n c u b a t e d tubes. T h i s is p o s s i b l y due to l y s i s of the cells such t h a t the released p r o t e i n was removed d u r i n g subsequent washings. T h e percent I C G for each test s a m p l e at each c o n c e n t r a t i o n (based o n c a l c u l a t i o n s u t i l i z i n g the m e a n p r o t e i n values for each t r e a t m e n t c o n d i t i o n ) appears t o i n d i c a t e t h a t the l i q u i d M A P a n d extracts of the h i g h crosslinked s o l i d s a m ple h a d essentially n o effect o n the g r o w t h o f L 9 2 9 cells i n t h i s assay. T h e l i q u i d e n z y m e at 4,000 μ g / 2 0 m L caused a 125.9% I C G . W h e n the l i q u i d e n z y m e was tested at lower concentrations ( f r o m 1 μg/20 m L ) , the percent I C G r a n g e d f r o m 40.3 t o 7 3 . 8 % . A l t h o u g h the concentrations of l i q u i d e n z y m e t h a t were tested i n t h i s e x p e r i m e n t d i d not p r o v i d e sufficient d a t a t o generate a n apparent dose response curve (absence of test concentrations between 4,000 a n d 500 i n the e x p e r i m e n t a l design), t h i s e n z y m e i n h i b i t s the g r o w t h o f L 9 2 9 cells. E x t r a c t s of the l o w crosslinked s o l i d also caused i n h i b i t i o n of cell g r o w t h i n t h i s assay. T h e percent I C G of the e x t r a c t s r a n g e d f r o m 15.4 to 5 9 . 3 % . A g a i n , no dose response was apparent w i t h the extracts of the low crosslinked s o l i d . T h e h i g h crosslinked s o l i d increased the p r o t e i n content of L 9 2 9 cells after a 7 2 - h r exposure i n a concentration-dependent m a n n e r . T h i s increase m i g h t be e x p l a i n e d either by a g r o w t h - e n h a n c i n g effect of the test m a t e r i a l or b y the c o n t r i b u t i o n of the test p r o t e i n m a t e r i a l itself. T h e low crosslinked s o l i d decreased the p r o t e i n content of L 9 2 9 cells after a 72-hr exposure p e r i o d i n a n inverse c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . T h i s finding is s t a t i s t i c a l l y significant at the 0.001 level b u t influenced h e a v i l y b y o n l y one single set of d a t a p o i n t s at the

33.


PicciANO


Mussels

479

highest e x t r a c t c o n c e n t r a t i o n (4,000 μ g / 2 0 m L ) . T h e l i q u i d e n z y m e decreased the p r o t e i n content of L 9 2 9 cells after a 72-hr exposure i n a c o n c e n t r a t i o n dependent m a n n e r a n d was c o m p a r a b l e i n t o x i c i t y at the highest c o n c e n t r a t i o n t o the p o s i t i v e c o n t r o l .

T a b l e I V . Percent I n h i b i t i o n of C e l l G r o w t h (% I C G ) Sample

Extract 0/g/20 mL)

Liquid M A P

L i q u i d enzyme

High Crosslinked

Low Crosslinked

4,000 500 100 50 4

-13.7 3 -0.7 2 4.4 1 0.2 4,000 125.9 500 57.6 100 48.1 50 70.7 4 40.3 3 73.8 2 61.1 1 58.1 14.4 4,000 500 18.8 100 -15.4 50 17.4 4 -8.1 3 -11.0 2 -30.1 1 -20.7 4,000 15.4 500 31.5 100 53.1 50 44.9 4 59.3 3 49.7 2 36.4 1

Negative control Positive control

ICG (%) -6.4 -5.2 10.1 -3.2

40.2 0 119.9

480


Discussion T h e p o t e n t i a l u t i l i t y a n d b i o c o m p a t i b i l i t y of M A P - b a s e d adhesives have been addressed i n the series of e x p e r i m e n t s presented here. M A P f o r m u l a t e d as C e l l T a k adhesive is a n efficient m e d i a t o r of m a m m a l i a n , b a c t e r i a l , a n d yeast cell a t t a c h m e n t . It has also been d e m o n s t r a t e d to s u p p o r t n o r m a l cell g r o w t h in vitro. M A P f o r m u l a t e d w i t h a crosslink c a t a l y s t is capable of b o n d i n g tissue t o tissue as d e m o n s t r a t e d w i t h corneal s t r o m a a n d b o n d i n g a l l o p l a s t i c m a t e r i a l s t o tissues, as s h o w n i n the b o n d i n g of a h y d r o g e l t o corneal s t r o m a . M o r e o v e r , two adhesive f o r m u l a t i o n s of M A P w i t h catechol oxidase at different r a t i o s were s h o w n t o be b i o c o m p a t i b l e i n a cell c u l t u r e agar overlay s y s t e m . N u m e r o u s cell types a t t a c h efficiently a n d r a p i d l y t o M A P . A n c h o r a g e dependent m a m m a l i a n cells, B C E , a n d B H K - 2 1 cells n o r m a l l y a t t a c h t o p l a s t i c a n d tissue c u l t u r e p l a s t i c w a r e coated w i t h cell c u l t u r e a t t a c h m e n t factors. O n c e a t t a c h e d , cells flatten a n d spread as a prerequisite for n o r m a l m e t a b o l i s m a n d g r o w t h . I n r e a l i t y , w i t h the m a n i p u l a t i o n s r e q u i r e d to s u b c u l t u r e or seed cells, o n l y a c e r t a i n percentage of the p o p u l a t i o n successfully completes the process. T h o s e t h a t do not a t t a c h a n d flatten q u i c k l y frequently do not s u r v i v e . T h i s is e s p e c i a l l y true for n e w l y i s o l a t e d cells f r o m a v a r i e t y of tissues, a l t h o u g h i t also applies t o established cultures. C e l l - T a k adhesive p r o m o t e d r a p i d a t t a c h ment of cells (greater t h a n 90%) seeded a n d , therefore, suggests the successful recovery o f a h i g h percentage of the p o p u l a t i o n . N u m e r o u s other cell types a n d p r i m a r y isolates (5) have been s t u d i e d w i t h successful i s o l a t i o n a n d s u b c u l t u r e o n C e l l - T a k p r o t e i n w i t h m a i n t e n a n c e of n o r m a l cell f u n c t i o n a n d m o r p h o l o g y . A n c h o r a g e - i n d e p e n d e n t cells grow as spheres i n suspension i n tissue c u l t u r e b r o t h a n d are not dependent o n the flattening a n d s p r e a d i n g m e c h a n i s m for n o r m a l cell g r o w t h t o occur. Efficiency of recovery of these cells is d e t e r m i n e d b y the density of C e l l - T a k adhesive. T h e a m o u n t used here, 5 / i g / c m , is sufficient for 8 5 % cell recovery. F o r cells t h a t flatten, lower densities are sufficient ( d a t a not s h o w n ) . C e l l s t h a t r e m a i n as spheres present a m u c h s m a l l e r surface area t o the adhesive. 2

T h e purpose for i m m o b i l i z i n g anchorage-independent cells, such as the U 937 h u m a n h i s t i o c y t i c l y m p h o m a l i n e s t u d i e d here, is not for enhanced recovery or for g r o w t h . R a t h e r , e x p e r i m e n t a l m a n i p u l a t i o n s t h a t are most r e a d i l y done o n i m m o b i l i z e d cells c a n now be performed o n these a n d other suspension cells. E x a m p l e s i n c l u d e the v a r i o u s i m m u n e assays. A specific e x a m p l e is a n assay where the efficient recovery o f a l l cells (such as T - c e l l s ) is c r i t i c a l . T - c e l l s n o r m a l l y do n o t a t t a c h t o glass slides or tissue c u l t u r e p l a s t i c w a r e . T h e a b i l i t y t o q u a n t i f y the relative p r o p o r t i o n s of T - c e l l s u b p o p u l a t i o n s w o u l d enable the r a p i d diagnosis of the i m m u n o l o g i c a l competence of p a t i e n t s u n d e r g o i n g cancer t h e r a p y or w i t h specific i m m u n e - s y s t e m diseases. A n o t h e r e x a m p l e of u t i l i t y is i n the studies of cell-cell interactions between p o p u l a t i o n s o f n o n a d h e r i n g cells. W i t h the a b i l i t y t o i m m o b i l i z e one of the two p o p u l a t i o n s (or two of three, etc.),

33.


PicciANO


Mussels

481

the p o p u l a t i o n s c a n be easily separated after a n i n c u b a t i o n p e r i o d together, a n d d y n a m i c i n t e r a c t i v e effects c a n be a n a l y z e d . Since U - 9 3 7 cells q u i c k l y a t t a c h w i t h h i g h efficiency t o C e l l - T a k , t h i s a p p r o a c h t o i m m o b i l i z i n g cells appears very p r o m i s i n g . T h e k i n e t i c s of a t t a c h m e n t for the diverse cell types s t u d i e d here were i d e n t i c a l . T h e c o m p o s i t i o n of M A P i n C e l l - T a k adhesive is w e l l u n d e r s t o o d . A s m u c h as 6 0 % of the h i g h m o l e c u l a r weight p r o t e i n is h y d r o x y l a t e d , a n d 2 0 % is l y s i n e , w h i c h c o n t r i b u t e s t o a large net p o s i t i v e charge. T h e p r o t e i n is a r e p e a t i n g p o l y m e r o f p e p t i d e s , the sequences o f w h i c h are k n o w n (6) a n d are d i s s i m i l a r f r o m the specific r e c o g n i t i o n site o n fibronectin (7,8). W e suggest t h a t the efficient a t t a c h m e n t of m a m m a l i a n cell types, w i t h the same k i n e t i c s , p l u s the i m m o b i l i z a t i o n of b a c t e r i a , b o t h g r a m p o s i t i v e a n d negative, a n d a yeast c o u l d o n l y be m e d i a t e d b y a nonspecific m e c h a n i s m c o m p r i s e d of e n t a n g l e m e n t , h y d r o g e n b o n d i n g , a n d i o n i c i n t e r a c t i o n s . T h e role of L - d o p a i n t h i s s y s t e m ( 1 0 - 1 5 % of the a m i n o a c i d c o m p o s i t i o n ) cannot be i g n o r e d . W h i l e these groups are capable of h y d r o g e n b o n d i n g t h r o u g h the double h y d r o x y l groups, t h e i r p o s i t i o n a n d o r i e n t a t i o n enables c h e l a t i o n t h r o u g h t r i v a l e n t a n d tetravalent ions. It is w e l l k n o w n t h a t L - d o p a is a h i g h l y efficient scavenger of ions (9). U n l i k e cell c u l t u r e , w o u n d r e p a i r requires at least a t w o - p a r t adhesive syst e m : M A P p l u s a c r o s s l i n k i n g agent. T h e issue of b i o c o m p a t i b i l i t y is inherent i n the use of M A P i n C e l l - T a k adhesive. T h r o u g h o u t these e x p e r i m e n t s , l i v i n g cells were m a i n t a i n e d i n the presence of M A P . T h e i n t r o d u c t i o n of the c r o s s l i n k i n g c o m p o n e n t i n t o the s y s t e m r e q u i r e d a réévaluation of c o m p a t i b i l i t y at the cell c u l t u r e level. T h i s was done u s i n g s t a n d a r d i z e d assays developed for the e v a l u a t i o n of the b i o c o m p a t i b i l i t y of m e d i c a l devices. It is a p p a r e n t t h a t M A P alone d i d n o t interfere w i t h cell g r o w t h or cause d a m a g e t o cells i n either assay. T h e c o m p o n e n t s of h i g h or low crosslinked M A P t h a t diffused t h r o u g h agar were not f o u n d t o be c y t o t o x i c . T h e catechol oxidase e n z y m e alone d i d affect cells i n c u l t u r e i n b o t h assays. T h e water e x t r a c t o f l o w - r a t i o c r o s s l i n k e d M A P also i n h i b i t e d cell g r o w t h b u t to a lesser degree. T h e catechol oxidase catalyzes the h y d r o x y l a t i o n of tyrosine t o L - d o p a a n d the conversion o f L - d o p a t o a q u i n o n e . T h e p o s s i b i l i t y exists t h a t e n z y m e , i n c o m p l e t e l y i m m o b i l i z e d or i n a c t i v a t e d w i t h i n the adhesive m a t r i x , c o u l d c a t a l y z e the reactions o n cell surface p r o t e i n s , t h e r e b y d a m a g i n g the cells. W i t h careful use of f o r m u l a t i o n s b l e n d e d at higher crosslink r a t i o s , t h i s s i t u a t i o n c a n be a v o i d e d . T h e development of m e t h o d s for the use of M A P i n w o u n d r e p a i r r e q u i r e d two p a r a l l e l sets of definitions. T h e first i n v o l v e d M A P i t s e l f w i t h the i n v e s t i g a t i o n o f c o n c e n t r a t i o n s o f p r o t e i n a n d ratios o f c r o s s l i n k i n g c a t a l y s t t h a t y i e l d e d sufficient b o n d strengths. T h e second was the choice of m o d e l systems a n d the a n a l y s i s of tissue-related parameters t h a t i m p i n g e d o n adhesion t e s t i n g . T h e c o m p l e x i t y of tissues necessitated t h a t s p e c i a l a t t e n t i o n be g i v e n t o m a n y details so t h a t reliable test d a t a c o u l d be o b t a i n e d . T w o test systems were used here. W i t h the development of the o v e r l a p p i n g corneal b o n d s y s t e m , i t was learned t h a t the e p i t h e l i a l a n d e n d o t h e l i a l cell l a y -

482


ers were a w e a k l y a d h e r i n g surface layer o n the corneal s t r o m a a n d h a d t o be removed p r i o r t o t e s t i n g i n order t o m a x i m i z e j o i n t strengths. T h e s t r o m a of enucleated eyes begins t o decompose a n d weaken w i t h t i m e , even w i t h storage at 4 ° C a n d , therefore, h a d t o be used o n the day of slaughter. T h e c u r v a t u r e of the cornea h a d t o be reduced as m u c h as possible t o p e r m i t close a p p o s i t i o n o f the tissues. T h i s was a c c o m p l i s h e d b y t r i m m i n g the c u r v e d cornea i n t o s t r i p s , w h i c h were t h e n used for t e s t i n g . E v e n w i t h these measures, the d a t a were not always consistent. T r e n d s were seen w i t h a r e q u i r e m e n t for i n creased crosslink c a t a l y s t r a t i o s w i t h increased p r o t e i n a m o u n t s , suggesting t h a t higher cohesive s t r e n g t h is r e q u i r e d w i t h increased p r o t e i n . However, i n t h i s s y s t e m , increased M A P p r o t e i n d i d not necessarily y i e l d higher b o n d strengths. L o w e r t o t a l p r o t e i n a m o u n t s (50 / / g / c m ) consistently y i e l d e d the highest b o n d strengths. Increased p r o t e i n c o u l d be r e t a r d i n g close tissue a p p o s i t i o n b y creat i n g a n u n d e s i r a b l e b o n d d e p t h t h a t i n t u r n w o u l d require increased c r o s s l i n k i n g for increased cohesive s t r e n g t h . 2

T h e s e a l i n g o f corneal perforations u s i n g w h o l e enucleated b o v i n e eyes re q u i r e d the same a t t e n t i o n t o tissue p r e p a r a t i o n . T h e e p i t h e l i a l layer i n the b o n d area was r e m o v e d . A f t e r the p u n c t u r e of the cornea, the s t r o m a was r i n s e d a n d b l o t t e d t o remove p o t e n t i a l l y interfering aqueous h u m o r c o m p o n e n t s . T h e H y p a n h y d r o g e l m a t e r i a l , w h i c h is 8 0 - 9 0 % w a t e r , was r o u t i n e l y presoaked i n P B S . M a x i m u m b o n d strengths u s i n g 53 μg of M A P per c m p a t c h were o b t a i n e d i n 10 m i n after a p p l i c a t i o n . 2

Several other investigators have c o n d u c t e d e x p e r i m e n t s t o address d y n a m i c aspects of M A P f o r m u l a t i o n s used i n our studies. T h e adhesive has been f o u n d t o be freely p e r m e a b l e t o i n u l i n , w h i c h has a m o l e c u l a r weight of 42,000 (10), at the e n z y m e t o M A P r a t i o of 12:1 (12 Uy^/g M A P p r o t e i n ) w i t h u p t o 200 m g of t o t a l p r o t e i n per c m . T h i s suggests t h a t , at t h i s c o n c e n t r a t i o n , M A P does not f o r m a s o l i d film a n d t h a t corneal cells a n d tissues w o u l d not be d a m a g e d due to r e t a r d e d gas or n u t r i e n t p e r m e a b i l i t y . T h e p o t e n t i a l exists for w o u n d h e a l i n g to occur not o n l y a r o u n d b u t also t h r o u g h the adhesive m a t r i x . A d d i t i o n a l l y , at the crosslink c a t a l y s t r a t i o used here a n d a higher r a t i o of 15:1, the adhesive m a t r i x r e m a i n s susceptible t o p r o t e o l y t i c digestion ( T w i n i n g , p e r s o n a l c o m m u n i c a t i o n ) . T h e rate of digestion has not yet been e s t a b l i s h e d . It is a n t i c i p a t e d t h a t the rate is dependent o n degree of c r o s s l i n k i n g a n d , therefore, t h a t f o r m u l a t i o n s c o u l d be t a i l o r e d to meet h e a l i n g rate requirements of diverse tissues. S t u d i e s have b e g u n t h a t address t h i s issue in vivo i n r a b b i t s . P r e l i m i n a r y results show t h a t b o t h the corneal overlap a n d p e r f o r a t i o n sealant m o d e l s developed here in vitro are feasible in vivo. D o n o r a n d recipient corneas were b o n d e d w i t h M A P adhesive i n c o n j u n c t i o n w i t h a m i n i m u m n u m b e r of t e m p o r a r y sutures. B o t h corneas r e m a i n e d clear a n d f u n c t i o n e d n o r m a l l y . W i t h large (4 m m ) perforations of r a b b i t corneas, the H y p a n h y d r o g e l p l u s M A P adhesive s y s t e m f a c i l i t a t e d the f o r m a t i o n of a s o l i d p l u g w i t h i n 24-hr postsurgery. W i t h the e x c e p t i o n of m i n i m a l i n f l a m m a t i o n due to the i n j u r y i n d u c e d e x p e r i m e n t a l l y , the eyes appeared n o r m a l , a n d there was no leakage at the p l u g site. H i s t o p a t h o l o g y 2

33.


PicciANO


Mussels

483

o n subsequent days f o l l o w i n g repair o f the eyes showed t h a t t h e p l u g appeared fibrous, b u t t h e n a t u r e o f t h e m a t e r i a l is y e t t o be e s t a b l i s h e d . N o n e o f t h e a n i m a l s showed a n y tissue d a m a g e as a result o f the adhesive. Conclusions N u m e r o u s studies are p l a n n e d o r i n progress t o f u r t h e r e s t a b l i s h t h e efficacy of M A P for use i n m e d i c a l a n d s u r g i c a l a p p l i c a t i o n s . Tissues other t h a n those described here m a y have special requirements necessitating t a i l o r i n g o f the for m u l a t i o n s . A l t e r n a t e m e c h a n i s m s for c r o s s l i n k i n g a n d t h e a d d i t i o n o f fillers are b e i n g i n v e s t i g a t e d . W e have n o t yet f u l l y achieved a l l t h e characteristics o f the m u s s e l - p r o d u c e d adhesive, b u t t h e f o r m u l a t i o n s a n d delivery systems used here show great p o t e n t i a l i n soft tissue w o u n d r e p a i r . Acknowledgment s T h e a u t h o r s g r a t e f u l l y acknowledge the t e c h n i c a l assistance o f T i m o t h y H o u s l e y , E l s i e M a t h e w s , a n d R i c h K u s l e i k a . W e also t h a n k L e e - A n n C a r r o l l a n d J u l i a n n e S a l i c k i for t h e care t h e y t o o k i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . Literature

Cited

1. Waite, J. H.; Tanzer, M. L. Science, 1981, 212, 1038-1040. 2. Waite, J. H. J. Biol Chem., 1983, 258, 2911-2915. 3. Waite, J. H. U.S. Patent 4 585 585, 1986. 4. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem., 1951, 193, 265-275. 5. Munoz-Blay, T.; Benedict, C. V.; Picciano, P. T.; Cohen, S. J. Exp. Pathol., in press. 6. Waite, J. H.; Housley, T. J.; Tanzer, M. L. Biochem., 1985, 24, 5010-5014. 7. Pierschbacher, M. D.; Ruoslahti, E . Nature, 1984, 309, 30-33. 8. Yamada, Κ. M.; Kennedy, D. W. J. CellBiol.,1984, 99, 29-36. 9. Avdeef, Α.; Sofen, S. R.; Bregante, T. L.; Raymond, Κ. N. J. Am. Chem.Soc.,1978, 100, 5362-5370. 10. Green, K.; Berdecia, R.; Cheeks, L. Current Eye Research, 1987, 6, in press. RECEIVED May 24, 1988

Adhesives from Marine Mussels

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