Hydrogels for Medical and Related Applications - ACS Publications

24 Fibrous Capsule Formation and Fibroblast Interactions at Charged Hydrogel Interfaces J. J. ROSEN and D. F. GIBBONS Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106

Downloaded by UNIV OF ALBERTA on June 7, 2013 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0031.ch024

L. A. CULP Department of Microbiology, Case Western Reserve University, Cleveland, Ohio 44106

Precise measurement of the biological response to specific properties of an implanted material requires a well defined materials system and a method for accurately evaluating variations in the response. Ionogenic hydrogels have been used as a materials system in this study to evaluate the effect of charge on the early phases of fibrous capsule formation. Image analysis techniques were used to quantitatively measure the response. Specific aspects of fibroblast interactions with these materials were also investigated using cell culture techniques. The fibrous capsule is a layer of connective tissue formed as part of the biological response to an implanted foreign material. It consists primarily of collagen fibers, but may also contain a variety of inflammatory cell types and new capillaries, depending on the degree of the response(1). It has been previously demonstrated in vivo, that the addition of charged functional groups to a hydrogel network can significantly alter the early phases of the response (2). An understanding of the mechanisms responsible for these changes is an essential step in the development of materials which can fulfill the varied requirements encountered in soft-tissue implant applications. Although many attempts have been made to correlate cell attachment with properties of the substrate material in culture (.3 they have generally been limited by an ability to uniquely vary the specific material parameter of interest. The hydrophilic gel polymers based on hydroxyethyl methacrylate (HEMA) represent a class of well-characterized materials which are particularly well suited to isolate the effect of surface charge on cell attachment kinetics. The same hydrogel compositions were used in the implant study and for cell culture substrates. This allowed direct comparisons to be made between the effect of surface charge on 329

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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t h e i n v i v o r e a c t i o n , and on t h e c e l l u l a r r e s p o n s e in vitro.

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Methods and M a t e r i a l s Monomers. 2 - H y d r o x y e t h y l m e t h a c r y l a t e as r e c e i v e d f r o m P o l y s c i e n c e s , I n c . , c o n t a i n e d between 0.25 and 0 . 4 0 p e r c e n t m e t h a c r y l i c a c i d as an i m p u r i t y , 2 0 0 - 4 0 0 ppm MEHQ as an i n h i b i t o r and had a pH o f 6.00 t o 6 . 2 0 . Adsorption with potassium carbonate (anhydrous) lowered the m e t h a c r y l i c a c i d c o n t e n t t o b e l o w 0.15 p e r c e n t and r e d u c e d t h e i n h i b i t o r s l i g h t l y t o 1 5 0 - 2 0 0 ppm. The monomer p u r i t y l e v e l s were d e t e r m i n e d u s i n g gas c h r o m a t o g r a p h y . The f i n a l pH was 6.85 t o 6 . 9 0 . Methacrylic a c i d ( P o l y s c i e n c e s , I n c . ) ( b . p . = 5 9 ° C , 10mm Hg.) and d i e t h y l ami no e t h y l m e t h a c r y l a t e (Rohm and Haas C o . ) ( b . p . = 9 0 ° C , 3 mm Hg.) were vacuum d i s t i l l e d u n d e r d r y n i t r o g e n and t h e i r f i n a l p u r i t y was c h e c k e d u s i n g gas c h r o m a t o g r a p h y . Tetrae t h y l e n e g l y c o l d i m e t h a c r y l a t e ( P o l y s c i e n c e s , I n c . ) was t h e c r o s s l i n k i n g a g e n t and was u s e d as r e c e i v e d . Polymerization. S h e e t s o f h y d r o p h i l i c g e l s were p r e p a r e d by s o l u t i o n p o l y m e r i z a t i o n between g l a s s p l a t e s w i t h 1.0 m i l l i m e t e r g l a s s s p a c e r s f o r t h e i m p l a n t m a t e r i a l s and 0.15 m i l l i m e t e r g l a s s spacers f o r the c e l l c u l t u r e s u b s t r a t e s . P o l y m e r i z a t i o n p r o c e e d e d f o r 24 h o u r s a t room t e m p e r a t u r e . The r e a c t i o n mix c o m p o s i t i o n s a r e g i v e n i n T a b l e I. The g e l s were removed f r o m t h e g l a s s by s o a k i n g i n d i s t i l l e d w a t e r . All h y d r o g e l s were washed i n d i s t i l l e d w a t e r w h i c h was changed d a i l y , f o r a minimum o f t h r e e w e e k s . E q u i l i b r i u m water content o f t h e g e l s was d e t e r m i n e d on f u l l y h y d r a t e d s a m p l e s by m e a s u r i n g t h e p e r c e n t w e i g h t l o s s a f t e r vacuum d r y i n g a t 120°C u n t i l no f u r t h e r w e i g h t l o s s was o b s e r v e d . In V i v o . I m p l a n t s p e c i m e n s , m e a s u r i n g 7 χ 15 mm., were c u t f r o m t h e washed g e l s . The s a m p l e s were r e e q u i l i b r a t e d i n p h o s p h a t e b u f f e r e d s a l i n e s o l u t i o n f o r t h r e e days and b o i l e d f o r 30 m i n u t e s p r i o r t o i m p l a n t a t i o n . S p r a g u e - D a w l e y male r a t s , w e i g h i n g between 300 and 350 grams were used f o r a l l e x p e r i m e n t s . S u b c u t a n e o u s i m p l a n t s were p l a c e d away f r o m t h e i n c i s i o n i n a p o c k e t opened p r e c i s e l y a t t h e f a c i a l a y e r o f t h e d o r s a l s u r f a c e . The i m p l a n t s were removed w i t h t h e s u r r o u n d i n g t i s s u e s a f t e r v a r i o u s t i m e s f r o m one t o t w e n t y - o n e d a y s , f i x e d i n f o r m a l i n and p r e p a r e d f o r h i s t o l o g y by s t a n d a r d t e c h n i q u e s . Sham e x p e r i ments i n w h i c h t h e e n t i r e s u r g i c a l p r o t o c o l was p e r f o r m e d , b u t w i t h no m a t e r i a l i m p l a n t e d , s e r v e d as c o n t r o l s . The t i s s u e r e s p o n s e was e v a l u a t e d q u a l i t a t i v e l y on t h e b a s i s o f c e l l t y p e s , d e g r e e o f g r a n u l a t i o n , m o r p h o l o g y o f c o l l a g e n o r g a n i z a t i o n and i n t e g r i t y of implant m a t e r i a l . A modified tri-chrome stain d i f f e r e n t i a t e d t h e c o l l a g e n f i b e r s f r o m s u r r o u n d i n g f e a t u r e s and provided the b a s i s f o r q u a n t i t a t i v e a n a l y s i s of the f i b r o u s c a p s u l e . C o m p u t o r - a s s i s t e d image a n a l y s i s u s i n g a M i l l i p o r e p a r t i c l e c o u n t i n g m i c r o s c o p e was u s e d t o measure t h e mean



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d i s t a n c e i n m i c r o n s f r o m t h e m a t e r i a l i n t e r f a c e t o t h e edge o f the f i b r o u s c a p s u l e , t h e percentage o f t h e capsule c r o s s s e c t i o n a l area a c t u a l l y f i l l e d w i t h c o l l a g e n f i b e r s , and t h e t o t a l c o l l a g e n area i n square microns p e r u n i t l e n g t h along t h e m a t e r i a l i n t e r f a c e . A m o d i f i e d n u c l e a r s t a i n was u s e d t o c o u n t t h e number o f c e l l s p r e s e n t p e r u n i t a r e a a d j a c e n t t o t h e m a t e r i a l i n t e r f a c e . A l l measurements were made a t s e v e n l o c a t i o n s a d j a c e n t t o t h e i n t e r i o r s u r f a c e o f t h e i m p l a n t , and were r e p e a t e d on two s e c t i o n s f o r e a c h m a t e r i a l a n d e a c h i m p l a n t time. The r e s u l t s a r e r e p o r t e d as t h e mean o f t h e s e m e a s u r e ments. In V i t r o . A l l c e l l c u l t u r e e x p e r i m e n t s were p e r f o r m e d u s i n g t h e S w i s s 3T3 c e l l l i n e , c o n t a c t i n h i b i t e d mouse f i b r o blasts. O n l y c e l l s between t h e i r 1 0 t h and 2 0 t h p a s s a g e were used f o r t h i s s t u d y . C e l l s were r o u t i n e l y grown i n E a g l e s ' m i n i m a l e s s e n t i a l medium w h i c h was s u p p l e m e n t e d w i t h f o u r t i m e s t h e normal c o n c e n t r a t i o n o f amino a c i d s and v i t a m i n s (MEMX4), p e n i c i l l i n ( 2 5 0 u n i t s / m l ) , s t r e p t o m y c i n ( 0 . 2 5 m g / m l ) , and 10 p e r c e n t d o n o r c a l f s e r u m . The DNA o f t h e s o u r c e c e l l s was l a b e l l e d w i t h ^ H - t h y m i d i n e by g r o w i n g c e l l s f o r 24 h o u r s i n media c o n t a i n i n g 0.1 y C i / m l o f H - t h y m i d i n e f o l l o w e d by 48 h o u r s o f g r o w t h i n n o n - l a b e l l e d media ( 6 ) . T h i s t e c h n i q u e p r o v i d e d a method f o r c o u n t i n g t h e c e l l s a n d d e t e r m i n i n g t h e r e l a t i v e a b i l i t y o f S w i s s 3T3 f i b r o b l a s t s t o a t t a c h t o s u b s t r a t e s o f v a r i o u s hydrogel compositions. The s u b s t r a t e s were p r e p a r e d f r o m washed h y d r o g e l membranes e q u i l i b r a t e d i n p h o s phate b u f f e r e d s a l i n e . D i s c s ( 1 5 mm d i a m e t e r ) o f t h e g e l s were p l a c e d i n t h e a t t a c h m e n t media f o r two h o u r s p r i o r t o t h e attachment assay. The a t t a c h m e n t media c o n t a i n e d p h o s p h a t e b u f f e r e d s a l i n e s u p p l e m e n t e d w i t h d i v a l e n t c a t i o n s ( 1 0 0 mg/1 M g S 0 £ . 7 H 0 and 100 mg/1 C a C l 2 ) and 10% d o n o r c a l f s e r u m . 3

2

The a t t a c h m e n t a s s a y c o n s i s t e d o f i n o c c u l a t i n g d i s h e s c o n t a i n i n g h y d r o g e l s u b s t r a t e s w i t h a known a l i q u o t ( 2 . 0 χ 10 c e l l s i n one m i l l i l i t e r o f a t t a c h m e n t m e d i a ) o f t h e l a b e l l e d cells. A t e a c h o f t h e t i m e p o i n t s , one d i s c was removed f r o m each d i s h ( t h e r e were two d i s h e s f o r e a c h g e l c o m p o s i t i o n ) , d i p p e d t h r e e t i m e s i n t h r e e s e p a r a t e washes o f p h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g d i v a l e n t c a t i o n s ( 1 0 0 mg/1 M g S O ^ r ^ O and 100 Mg/1 C a C l 2 ) and t h e n p l a c e d i n s c i n t i l l a t i o n v i a l s containing Bray's s c i n t i l l a t i o n solution. fi

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R e s u l t s and D i s c u s s i o n The b i o l o g i c a l r e a c t i o n t o t h e p r e s e n c e o f an i m p l a n t e d m a t e r i a l , o f t e n r e f e r r e d t o as t h e " f o r e i g n body r e s p o n s e " , i s i n f l u e n c e d by a c o m p l e x s e t o f v a r i a b l e s . A recent review ( 7 j d i f f e r e n t i a t e d some m a j o r c l a s s e s o f t h e s e v a r i a b l e s a n d a t t e m p t e d t o d i s t i n g u i s h between t h o s e a s s o c i a t e d w i t h m a t e r i a l p r o p e r t i e s a n d t h o s e i n v o l v e d i n t h e " n o r m a l " wound h e a l i n g process.



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The l a r g e number o f p o t e n t i a l l y r e l e v a n t f a c t o r s has sometimes made p r e v i o u s i n v e s t i g a t i o n s d i f f i c u l t t o c o n t r o l and t h e i r r e s u l t s d i f f i c u l t t o i n t e r p r e t . The i s s u e o f what c o n s t i t u t e s a " b i o c o m p a t i b l e " m a t e r i a l i n t h e s o f t t i s s u e s has been f u r t h e r c o n f u s e d by a p e r s i s t a n t n o t i o n t h a t a t h i n f i b r o u s membrane a r o u n d an i m p l a n t i s a l w a y s a " g o o d " r e a c t i o n and a t h i c k c a p s u l e i s i n d i c a t i v e o f a s e v e r e o r " b a d " r e s p o n s e . T h i s c o n c e p t was f u r t h e r e d by L a i n g ' s e a r l y s t u d y ( 8 ) o f i m p l a n t e d m e t a l s w h i c h d e m o n s t r a t e d t h a t an i n c r e a s e i n m e t a l i o n c o n c e n t r a t i o n o r i o n t o x i c i t y c o r r e l a t e d w i t h an i n c r e a s e i n t h e s u r r o u n d i n g f i b r o u s membrane t h i c k n e s s . Although i t i s true that chronic inflammation, t i s s u e n e c r o s i s , o r c o n t i n u e d c e l l u l a r p r o l i f e r a t i o n w o u l d be c o n s i d e r e d u n d e s i r a b l e i n a l m o s t any c l i n i c a l a p p l i c a t i o n o f an i m p l a n t e d m a t e r i a l , i t does n o t f o l l o w t h a t a s t a b l e , t h i c k f i b r o u s c a p s u l e p e r s e , w o u l d a l w a y s be an u n d e s i r a b l e c o n sequence o f t h e b i o l o g i c a l r e a c t i o n . F o r e x a m p l e , when m e c h a n i c a l s t a b i l i t y i s d e s i r e d t o l i m i t l a t e r a l movement, s u c h as i n a hydrocephalus shunt, a dense, well organized a c e l l u l a r , and n o n a d h e r e n t c a p s u l e w o u l d a l l o w f o r l o n g i t u d i n a l g r o w t h adjustments w h i l e p r o v i d i n g the necessary l a t e r a l s t a b i l i t y . In t h e same a p p l i c a t i o n , t h e f o r m a t i o n o f f i b r o u s t i s s u e i n o r a r o u n d t h e d r a i n s e c t i o n o f t e n l e a d s t o b l o c k a g e and e v e n t u a l d e v i c e f a i l u r e . A much b e t t e r u n d e r s t a n d i n g o f b a s i c i n t e r a c t i o n s between m a t e r i a l p a r a m e t e r s and b i o l o g i c a l m e d i a t o r s i s needed b e f o r e t h i s h i g h l e v e l o f c o n t r o l o v e r t h e r e a c t i o n can be a c h i e v e d . D u r i n g t h i s i n v e s t i g a t i o n o u r p r i m a r y o b j e c t i v e was t o determine i f the charge of the m a t e r i a l s i g n i f i c a n t l y a f f e c t e d t h e p r o c e s s o f f i b r o u s c a p s u l e f o r m a t i o n . The h y d r o g e l s y s t e m was s e l e c t e d as t h e m a t e r i a l model b e c a u s e t h e c h a r g e c o u l d be v a r i e d by c h a n g i n g t h e c h e m i c a l c o m p o s i t i o n o f t h e f u n c t i o n a l g r o u p s w i t h o u t c h a n g i n g t h e main c h a i n m e t h a c r y l a t e s t r u c t u r e . The i n i t i a l monomer r e a c t i o n s o l u t i o n s , as shown i n T a b l e I, a l l c o n t a i n e d t h e same p e r c e n t o f c r o s s l i n k i n g a g e n t and t h e same amount o f w a t e r . A f t e r s w e l l i n g t o e q u i l i b r i u m t h e h i g h l y a c i d i c g e l s c o n t a i n e d s l i g h t l y more w a t e r (47%) t h a n t h e most b a s i c g e l s ( 4 1 % ) . These d i f f e r e n c e s may have c o n t r i buted to the observed v a r i a t i o n s i n the b i o l o g i c a l response, but t h e y were c o n s i d e r e d t o be s m a l l when compared w i t h t h e w i d e r a n g e o f c h a r g e d e n s i t y and s i g n t h a t was p o s s i b l e w i t h t h i s system. Scanning microscopy o f the polymers r e v e a l e d f a i r l y u n i f o r m , smooth s u r f a c e s w i t h s i m i l a r m o r p h o l o g y f o r a l l compositions. I t i s now w e l l e s t a b l i s h e d (7) t h a t many o t h e r e x p e r i m e n t a l p a r a m e t e r s must a l s o be c o n s i d e r e d as p o s s i b l e sources of a r t i f a c t during the e v a l u a t i o n of implanted m a t e r i a l s . To m i n i m i z e t h e i n f l u e n c e o f i m p l a n t edge and shape e f f e c t s ( 9 ) , o n l y t h e t i s s u e a d j a c e n t t o t h e c e n t r a l r e g i o n o f i m p l a n t s was i n c l u d e d i n the a n a l y s i s . The i m p l a n t , i t s e l f , was p l a c e d i n



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a " p o c k e t " opened by b l u n t d i s s e c t i o n away f r o m t h e s i t e o f t h e initial incision. V a r i a t i o n s due t o a n a t o m i c a l l o c a t i o n were r e d u c e d by a l w a y s p l a c i n g t h e i m p l a n t d i r e c t l y o v e r t h e m u s c l e f a c i a i n a p p r o x i m a t e l y i d e n t i c a l p o s i t i o n s f o r each a n i m a l . By c h o o s i n g t h i s p a r t i c u l a r p l a c e m e n t f o r t h e i m p l a n t , new c o l l a g e n formed a s p a r t o f t h e f i b r o u s c a p s u l e was e a s i l y d i f f e r e n t i a t e d from t h e " n o r m a l " t i s s u e s . F i g u r e 4 shows e x a m p l e s o f t h e i n t e r f a c e between t h e i m p l a n t (on t h e l e f t ) and t h e s u r r o u n d i n g t i s s u e s . Within the fibrous capsule, the collagen fibers f i l l e d only part o f the t i s s u e space. The r e m a i n d e r o f t h e c r o s s - s e c t i o n a l c a p s u l e a r e a was f i l l e d w i t h c e l l s , f l u i d , o r s m a l l v e s s e l s . The p e r centage o f the capsule area a c t u a l l y f i l l e d w i t h c o l l a g e n , r e f e r r e d t o i n T a b l e s I I , I I I , and IV a s " C o l l a g e n D e n s i t y " v a r i e d w i t h i m p l a n t t i m e and h y d r o g e l c o m p o s i t i o n and was r e l a t e d t o t h e morphology o f t h e newly formed c o l l a g e n f i b e r s . The t h i c k n e s s o f t h e c a p s u l e m u l t i p l i e d by t h e c o l l a g e n d e n s i t y was p r o p o r t i o n a l t o t h e t o t a l c o l t a g e n p r o d u c e d p e r u n i t a r e a along t h e capsule c r o s s - s e c t i o n . T h i s v a l u e was o b t a i n e d f r o m a r e a measurements made on 176 m i c r o m e t e r segments o f c a p s u l e f o r a l l samples and i s r e p o r t e d a s " R e l a t i v e Amount C o l l a g e n " i n Tables I I , I I I , IV. This a r b i t r a r y b u t c o n s t a n t u n i t o f c a p s u l e l e n g t h r e p r e s e n t e d a p p r o x i m a t e l y one m i c r o s c o p i c f i e l d a t 400x magnification. A p a r a l l e l measurement o f t o t a l c e l l u l a r a c t i v i t y i n t h e v i c i n i t y o f t h e i m p l a n t was d e t e r m i n e d by c o u n t i n g t h e n u c l e i o f a l l c e l l s w i t h i n 250 m i c r o m e t e r s o f t h e i m p l a n t s u r f a c e and normalizing t o a constant unit area. T h i s c o u n t was n o t l i m i t e d t o t h e c a p s u l e r e g i o n b e c a u s e s i g n i f i c a n t v e s s e l d e v e l o p m e n t and other c e l l a c t i v i t y often occurred outside the actual fibrous capsule. The number o f c e l l s p e r u n i t a r e a i n normal c o n n e c t i v e t i s s u e i s r e l a t i v e l y l o w ( a s i n d i c a t e d by t h e s u r g i c a l c o n t r o l ) and t h e r e f o r e , t h e v a l u e f o r t o t a l c e l l u l a r a c t i v i t y i s representative o f c e l l s that are d i r e c t l y involved i n the response t o the implant m a t e r i a l . The e n t i r e r a n g e o f t h e b i o l o g i c a l r e s p o n s e , f o r a l l m a t e r i a l s t e s t e d , was w i t h i n t h e l i m i t s o f what h a s been c o n s i d e r e d " m i l d " ( 8 ) . C o n t r a r y t o p r e v i o u s work ( 2 ) none o f t h e hydrogel m a t e r i a l s c o n s i s t a n t l y e l i c i t e d a "gross purulent exudate". O b v i o u s i n f e c t i o n s o f t h i s t y p e o c c u r r e d r a r e l y and did not correlate with the material compositions. The i n v i v o e x p e r i m e n t s o f t h i s s t u d y have d e m o n s t r a t e d s i g n i f i c a n t and r e p r o d u c i b l e c h a n g e s i n b o t h t h e amount o f c o l l a g e n p r o d u c e d and i t s o r g a n i z a t i o n a l m o r p h o l o g y a s a d i r e c t r e s u l t o f v a r i a t i o n s i n t h e charge o f t h e hydrogels. The b a r g r a p h s i n F i g u r e s 1 , 2 , a n d 3 show, f o r e x a m p l e , t h a t t h e c a p s u l e formed around p o s i t i v e l y charged hydrogels d u r i n g t h e f i r s t t h r e e weeks a f t e r i m p l a n t a t i o n i s t h i c k e r t h a n the capsules around o t h e r h y d r o g e l s . This r e s u l t agrees w i t h


334


TABLE I COMPOSITION OF HYDROGEL MONOMER SOLUTIONS COMPOSITION (Volu me Percent) DEAEMA HEMA MAA

HYDROGEL MONOMER (Mole Percent)


100 HEMA

100.0

95 HEMA, 5 MAA 80 HEMA,20 MAA 60 HEMA,40 MAA

96.0 84.0 67.5

95 HEMA, 5 DEAEMA 80 HEMA,20 DEAEMA 60 HEMA,40 DEAEMA

94.0 75.5 51.0

95 HEMA,2.5 MAA,2.5 DEAEMA 80 HEMA, 10 MAA, 10 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA

94.5 78.5 57.0

4.0 16.0 32.5 6.0 24.5 49.0 1.8 7.0 14.5

3.6 14.5 28.5

HEMA= 2-Hydroxyethyl methacrylate, MAA= M e t h a c r y l i c a c i d , DEAEMA= Diethyl ami no ethyl methacrylate, Crosslinker= Tetraethylene g l y c o l dimethacrylate: 2.2 mole percent, D i s t i l l e d water: 33.0 volume percent, Initiator: (NH^nSOg: 0.5 mole percent Reducing Agent: m^^S 0.5 mole percent. 1

IN

TABLE I I VIVO RESPONSE TO HYDROGELS IMPLANTED FOR ONE DAY

HYDROGEL MONOMER (Mole Percent)

CAPSULE THICKNESS (Micrometers) MEAN (S.D.)

COLLAGEN DENSITY (Percent) MEAN (S.D.)

RELATIVE AMOUNT COLLAGEN MEAN (S.D.)

TOTAL CELLULAR ACTIVITY MEAN (S.D.)

100 HEMA

27.1 (11.9)

54.4 ( 8.4)

14.2 ( 6.2)

192.1 (35.6)

95 HEMA, 5 MAA 60 HEMA,40 MAA

27.8 ( 4.2) 41.8 ( 8.6)

48.9 (10.6) 60.9 ( 9.7)

14.9 ( 3.9) 24.0 ( 9.0)

190.0 (26.6) 133.9 (20.6)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA

63.8 (19.8) 77.1 ( 7.2)

68.4 ( 9.4) 55.9 (10.9)

42.4 (11.5) 36.7 (13.5)

207.3 (57.9) 361.7 (49.6)

95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA

37.8 (10.4) 19.2 ( 6.9)

48.9 (10.6) 60.9 ( 9.7)

17.1 (9.8) 11.5 ( 3.8)

223.1 (55.5) 227.4 (48.9)

31.7 (10.6)

34.1 (11.7)

67.5 (17.7)

SURGICAL SHAM 1. 2. 3. 4.

4

1

2

3

Percent o f f i b r o u s capsule composed o f c o l l a g e n f i b e r s . Area o f c o l l a g e n f i b e r s p r o p o r t i o n a l to new c o l l a g e n formation. Number o f c e l l s per u n i t area adjacent to implant. Values obtained from connective t i s s u e adjacent to s u r g i c a l l y prepared site.


implant

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TABLE I I I IN VIVO RESPONSE TO HYDROGELS IMPLANTED FOR ONE WEEK HYDROGEL MONOMER (Mole Percent)



100 HEMA

84.8 (11.5)

81.1 ( 5.8) 59.6 (15.1)

130.6 (26.7)


97.9 (16.3) 79.4 (20.5)

65.7 ( 7.8) 61.4 ( 8.4)

63.7 ( 8.4) 46.9 (13.2)

87.6 (14.3) 159.8 (22.7)

112.6 (23.5) 175.0 (15.1)

49.6 ( 8.7) 52.0 ( 7.9)

57.2 (10.8) 91.2 (11.3)

287.4 (25.9) 248.1 (39.8)

87.7 (16.6) 85.3 (18.1)

54.6 (14.8) 62.1 ( 5.1)

50.7 ( 8.9) 53.1 ( 7.3)

133.9 (25.5) 149.5 (26.2)

51.8 ( 7.1)

87.0 (11.9)

88.5 (17.8)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA 95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA SURGICAL SHAM



TABLE IV IN VIVO RESPONSE TO HYDROGELS IMPLANTED FOR THREE WEEKS HYDROGEL MONOMER (Mole Percent)





100 HEMA

94.1 (11.6)

77.4 ( 6.4)

68.0 (11.6)

143.3 (25.0)


75.6 ( 7.6) 50.2 ( 7.9)

67.6 ( 6.2) 79.2 ( 6.5)

50.9 ( 5.6) 39.8 ( 7.7)

71.2 ( 6.7) 81.2 (16.3)

130.9 (28.0) 143.2 (17.9)

59.2 ( 4.9) 60.4 ( 5.5)

65.7 ( 7.1) 77.0 (15.4)

148.6 (19.5) 178.3 (14.2)

64.2 (14.6) 70.6 ( 5.8)

62.3 (10.8) 85.1 ( 7.3)

54.7 (11.2) 60.0 ( 6.4)

99.9 (16.9) 116.7 (21.9)

36.4 (10.1)

61.9 (17.2)

54.9 (16.0)

95 HEMA, 5 DEAEMA 60 HEMA,40 DEAEMA 95 HEMA,2.5 MAA,2.5 DEAEMA 60 HEMA, 20 MAA, 20 DEAEMA SURGICAL SHAM


336


60 % HEMA

100 %-HEMA


4 0 % DEAEMA

6 0 % HEMA

60%

4 0 % MAA

2 0 % MAA 20%

HEMA DEAEMA

Bar configurations used in Figures 2, 2, and 3 to represent the indicated hydrogel compositions

DAYS

IMPLANT

TIME

Figure 1. The effect of hydrogel composition and implant time on the mean thickness of the fibrous capsule



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IMPLANT

DAYS

TIME

Figure 2. The effect of hydrogel composition and implant time on the percent of the fibrous capsule composed of collagen fibers

21 DAYS

IMPLANT

TIME

Figure 3. The effect of hydrogel composition and implant time on the relative amount of collagen produced during the formation of the fibrous capsule




Figure 4. Fibrous capsule adjacent to hydrogel implants (at left) three weeks after implantation, 4a. 60% HEMA-40% MAA; 4b. 60% HEMA-40% DEAEMA; 4c. 100% HEMA. The tissues were stained with hematoxilin and eosin (340X ).



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t h e q u a l i t a t i v e o b s e r v a t i o n s made by B a r v i c {2). Quantitative e v a l u a t i o n p r o v i d e d by t h e image a n a l y s i s t e c h n i q u e s w h i c h have been summarized h e r e and d e s c r i b e d i n d e t a i l ( 1 0 ) r e v e a l s t h a t although the t o t a l t h i c k n e s s i s g r e a t e r f o r p o s i t i v e l y charged g e l s , t h e d e n s i t y o f c o l l a g e n f i b e r s d e c r e a s e s and t h e a c t u a l amount o f c o l l a g e n p r o d u c e d r e m a i n s e s s e n t i a l l y c o n s t a n t d u r i n g t h e same t h r e e week p e r i o d . To b e g i n t o d i f f e r e n t i a t e between p o s s i b l e models t h a t c o u l d a c c o u n t f o r t h e s e c h a n g e s , f i b r o b l a s t - h y d r o g e l i n t e r a c t i o n s were s t u d i e d u n d e r more c o n t r o l l e d c o n d i t i o n s . C e l l c u l t u r e s were used t o i n v e s t i g a t e t h e k i n e t i c s o f f i b r o b l a s t a t t a c h m e n t t o hydrogel s u b s t r a t e s . S u b s t r a t e p o l y m e r c o m p o s i t i o n s were c h o s e n f r o m , and i d e n t i c a l t o t h o s e used f o r t h e i n v i v o e x p e r i m e n t s . O n l y some o f t h e s e c o m p o s i t i o n s have been i n c l u d e d i n t h i s p a p e r to i l l u s t r a t e t h e range o f t h e observed r e a c t i o n s . The r e s u l t s o f t h e c e l l c u l t u r e a t t a c h m e n t e x p e r i m e n t s a r e p r e s e n t e d i n F i g u r e s 5 , 6 , and 7 . The v a l u e s f o r a t t a c h m e n t o f c e l l s a r e r e p o r t e d as a percentage o f t h e t o t a l c e l l s a v a i l a b l e f o r attachment. The s u b s t r a t e s were e q u i l i b r a t e d i n p h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g d i v a l e n t c a t i o n s and c a l f serum (see Methods). Fresh s o l u t i o n s o f t h i s composition also served as t h e a t t a c h m e n t m e d i a . For the short duration o f these e x p e r i m e n t s , t h e a b s e n c e o f v i t a m i n s and amino a c i d s n o r m a l l y f o u n d i n g r o w t h media d i d n o t a f f e c t t h e r a t e o r e x t e n t o f c e l l attachment ( 1 1 ) . The s p e c i f i c r o l e o f t h e serum p r o t e i n s has n o t been e l a b o r a t e d d u r i n g t h i s phase o f t h e s t u d y , a l t h o u g h i t i s n o t u n r e a s o n a b l e t o assume t h a t t h e s e p r o t e i n s i n t e r a c t d i f f e r e n t l y w i t h hydrogel s u r f a c e s o f d i f f e r e n t charges. The p r e s e n c e o r a b s e n c e o f serum has been shown t o i n f l u e n c e c e l l a t t a c h m e n t i n o t h e r model s y s t e m s ( 1 2 ) a n d i s p r o b a b l y i n v o l v e d i n t h e mechanisms r e s p o n s i b l e f o r t h e o b s e r v e d v a r i a t i o n s i n t h e h y d r o gel system. The i n i t i a l o b j e c t i v e o f t h i s s t u d y has been t o determine i f f i b r o b l a s t s respond d i f f e r e n t l y t o s u b s t r a t e s o f d i f f e r e n t c h a r g e d e n s i t y a n d s i g n when t h e r e a r e no i n t e r a c t i o n s w i t h o t h e r c e l l t y p e s , complement o r c l o t t i n g p r o t e i n s . The g r a p h s i n F i g u r e s 5 , 6 , and 7 show t h a t f i b r o b l a s t s do a l t e r t h e i r a t t a c h m e n t b e h a v i o r when t h e c h a r g e o f t h e s u b s t r a t e is varied. These changes o c c u r d u r i n g t h r e e d i s t i n c t p h a s e s of t h e attachment process. The f i r s t phase b e g i n s when t h e c e l l s f i r s t come i n c o n t a c t w i t h t h e s u b s t r a t e m a t e r i a l and ends when c e l l s begin t o a t t a c h r a p i d l y . T h i s so c a l l e d " l a g " phase i s o f t e n i n t e r p r e t e d as a time o f c e l l "accomodation" o r " a d j u s t ment" t o t h e s u b s t r a t e . The s e c o n d phase i s a p e r i o d o f r a p i d a t t a c h m e n t c h a r a c t e r i z e d by a s t e e p e r s l o p e on t h e a t t a c h m e n t curve. The t h i r d phase i s t h e " p l a t e a u " o r " l e v e l i n g - o f f " phase when t h e r e a r e no f u r t h e r s i g n i f i c a n t i n c r e a s e s i n t h e total c e l l s attached. F o r t h e n e u t r a l g e l s t h a t c o n t a i n e d no c h a r g e g r o u p s , t h e l a g phase l a s t e d between f i v e and t e n m i n u t e s . The p e r i o d o f r a p i d a t t a c h m e n t was o v e r w i t h i n f o r t y - f i v e


340


Figures 5, 6, and 7 are the results of an assay to determine the effect of hydrogel substrate composition on the attachment of Swiss 3T3 fibroblasts. The cells are labelled with H-thymidine and inocculated into dishes containing the hydrogel substrates. After times varying from 5 min to 2 hr, substrate discs are removed, washed, and counted using scintillation techniques to determine the percent of available cells that attached to the substrates.


3

Ο 60HEMA - 40 DEAEMA ο Θ0ΗΕΜΑ - 20-DEAEMA • 10 OHEMA

-o ι

TIME Figure 5.

IN

MINUTES

Attachment of Swiss 3T3 fibroblasts to hydrogel substrates


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341

342



m i n u t e s and t h e p l a t e a u a t t a c h m e n t v a l u e was a p p r o x i m a t e l y t w e n t y - f i v e p e r c e n t o f t h e a v a i l a b l e c e l l s . As n e g a t i v e c h a r g e s a r e added t o t h e g e l n e t w o r k , t h e l a g phase r e m a i n s e s s e n t i a l l y u n c h a n g e d , b u t t h e s l o p e o f r a p i d a t t a c h m e n t and t h e p l a t e a u value decrease. If positive c h a r g e s a r e a d d e d , t h e l a g phase goes t o w a r d z e r o and t h e s l o p e o f r a p i d a t t a c h m e n t i n c r e a s e s dramatically. The p l a t e a u v a l u e i n c r e a s e s , b u t i s n o t a t t h e maximum p e r c e n t a t t a c h e d due t o some c e l l s r e l e a s i n g f r o m t h e s u b s t r a t e a t around t h i r t y minutes. The i o n o g e n i c n e u t r a l g e l s w h i c h c o n t a i n e q u i m o l a r amounts o f p o s i t i v e and n e g a t i v e c h a r g e g r o u p s , have p r o p o r t i o n a t e l y l o w e r v a l u e s f o r e a c h phase t h a n any o f t h e o t h e r m a t e r i a l s . Conclusions The i o n o g e n i c h y d r o g e l s have p r o v i d e d a u s e f u l model f o r e v a l u a t i n g t h e e f f e c t o f c h a r g e on t h e e a r l y p h a s e s o f t h e s o f t - t i s s u e response. Q u a n t i t a t i v e e v a l u a t i o n and c o m p a r i s o n o f t h e c a p s u l e t h i c k n e s s , c o l l a g e n d e n s i t y and t h e amount o f c o l l a g e n p r o d u c e d , and t h e r e l a t i v e d e g r e e o f c e l l u l a r a c t i v i t y were made p o s s i b l e by t h e image a n a l y s i s o f s p e c i a l l y p r e p a r e d h i s t o l o g y s e c t i o n s o f the i n v i v o response to the implanted hydrogels. The n e u t r a l , homopolymer o f h y d r o x y e t h y l m e t h a c r y l a t e i s p r o g r e s s i v e l y e n c a p s u l a t e d d u r i n g the f i r s t t h r e e weeks. The c a p s u l e c o n t a i n s i n c r e a s i n g amounts o f c o l l a g e n w h i c h pack more e f f i c i e n t l y w i t h time t o produce a moderate, d e n s e l y packed structure. As p o s i t i v e c h a r g e s a r e added by c o - p o l y m e r i z i n g with d i e t h y l aminoethyl m e t h a c r y l a t e , s i g n i f i c a n t q u a n t i t i e s of c o l l a g e n a r e p r o d u c e d i m m e d i a t e l y a f t e r i m p l a n t a t i o n and a r e a c c o m p a n i e d by v e r y h i g h c e l l u l a r a c t i v i t y . A l t h o u g h new c o l l a g e n c o n t i n u e s t o be p r o d u c e d , e f f i c i e n t p a c k i n g i s h i n d e r e d ( p e r h a p s by t h e h i g h c e l l a c t i v i t y ) , and v e r y t h i c k , l o o s e , capsules r e s u l t . The f a c t t h a t t h e c a p s u l e does n o t o r g a n i z e e f f i c i e n t l y w i t h time i s c h a r a c t e r i s t i c of the response to these m a t e r i a l s and i s i n d i c a t e d by t h e c o n s i s t a n t l y l o w v a l u e f o r t h e collagen density. F i g u r e 4b i s t y p i c a l o f t h e c a p s u l e formed around p o s i t i v e l y charged g e l s . The a c i d i c g r o u p s a l s o have i n i t i a l l y high c e l l a c t i v i t y , but t h i s l e v e l decreases d u r i n g t h e s e c o n d week t o a l l o w f o r dense c a p s u l e f o r m a t i o n w i t h o u t l a r g e q u a n t i t i e s of c o l l a g e n being produced. When e q u i m o l a r q u a n t i t i e s o f a c i d i c and b a s i c g r o u p s a r e present i n the g e l , the e a r l y c e l l a c t i v i t y decreases w i t h i n a f e w d a y s and r e m a i n s s i g n i f i c a n t l y l o w e r t h a n t h e n e u t r a l g e l throughout the response. The c o l l a g e n p r o d u c t i o n a r o u n d t h e s e g e l s d e v e l o p s g r a d u a l l y and r e m a i n s c o n s t a n t a t v a l u e s l e s s t h a n o r equal to the n e u t r a l g e l . The c e l l c u l t u r e a t t a c h m e n t a s s a y showed t h a t as t h e g e l s a r e changed f r o m h i g h l y b a s i c t h r o u g h n e u t r a l t o h i g h l y acidic: 1) the time f o r c e l l accomodation or " l a g phase" i s


24.

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Fibroblasts

343

increased, 2 ) t h e s l o p e o f t h e r a p i d a t t a c h m e n t phase d e c r e a s e s s h a r p l y , and 3 ) t h e maximum p e r c e n t o f a t t a c h e d c e l l s d e c r e a s e s . C e l l attachment t o ionogenic, e l e c t r o n e u t r a l gels i s p r o p o r t i o n a t e l y l o w e r t h a n f o r any o t h e r g e l s . The i m p l i c a t i o n s o f t h e c e l l c u l t u r e e x p e r i m e n t s c a n n o t be f u l l y a p p r e c i a t e d u n t i l f u r t h e r s t u d i e s w h i c h a r e d e s i g n e d t o e l a b o r a t e t h e mechanisms o f a d h e s i o n t o h y d r o g e l substrates a r e c o m p l e t e d . A l t h o u g h d i r e c t c o r r e l a t i o n between c e l l c u l t u r e * i n v i v o experiments i s premature a t t h i s t i m e , c e r t a i n i n t e r e s t i n g trends are apparent. For example, t h e p o s i t i v e l y c h a r g e d g e l s have a h i g h e r l e v e l o f c e l l a c t i v i t y a r o u n d i m p l a n t s and a much more r a p i d and e x t e n s i v e a t t a c h m e n t o f fibroblasts in culture. I f t h e subsequent r e l e a s e o f c e l l s f r o m p o s i t i v e l y c h a r g e d s u b s t r a t e s r e p r e s e n t s c e l l damage o r d e a t h and t h i s a l s o o c c u r s a t t h e i m p l a n t i n t e r f a c e , t h e l o w d e n s i t y o f c o l l a g e n i n t h e c a p s u l e may be r e l a t e d t o i n c r e a s e d numbers o f c e l l s and c e l l d e b r i s a s s o c i a t e d w i t h t h e i m p l a n t interface.


a n (

The combined i n f o r m a t i o n o b t a i n e d f r o m f u r t h e r c e l l c u l t u r e and q u a n t i t a t i v e a n a l y s i s o f i n v i v o r e a c t i o n s w i l l be u s e f u l i n e v a l u a t i n g t h e r o l e o f s p e c i f i c mechanisms and b i o l o g i c a l pathways t h a t c o n t r o l t h e f o r m a t i o n o f f i b r o u s c a p s u l e s a r o u n d implanted m a t e r i a l s .

Literature Cited 1.

Kellermeyer, R.W., and K.S. Warren, J . of Exp. Med., (1970), 131, (1) 21-39. 2. Barvic, M., J . Vacik, D. Lim, and M. Zavadil, J . of Biomed. Mat. Res. (1971) 5, 225-238. 3. Grinnell, F., M. Milam, and P.A. Srere, Arch. of Biochem. and Biophys., (1972), 153, 193-198. 4. Rappaport, C., in "The Chemistry of Biosurfaces", M.L. Hair ed., Marcel Dekker, Inc., New York (1972). 5. Weiss, L., Exper. Cell Res., (1974), 83, 311. 6. Culp, L.A., J . of Cell Biol. (1974), 63, 71-83. 7. Coleman, D.L., R.N. King, and J.D. Andrade, J . of Biomed. Mat. Res. (1974), 8, (5) 199. 8. Laing, P.G., A.E. Ferguson, and E.S. Hodge, J . of Biomed. Mat. Res. (1967), 1, 135. 9. Wood, N.K., E.J. Kaminski, and R.J. Oglesby, J . of Biomed. Mat. Res., (1970), 4, 1. 10. Rosen, J.J., and D.F. Gibbons, (submitted for publication). 11. Mapstone, R. and L.A. Culp, (submitted for publication). 12. Witkowski, J.A. and W.D. Brighton, Exper. Cell Res., (1972) 70, 41.


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