Collagen Surfaces in Biomedical Applications - Advances in

2 Collagen Surfaces in Biomedical Applications KURT H. STENZEL, TERUO MIYATA, ITARU KOHNO, SUSAN D. SCHLEAR, and ALBERT L. RUBIN

Downloaded by CORNELL UNIV on September 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch002

Rogosin Laboratories, The New York Hospital-Cornell Medical Center, Departments of Surgery and Biochemistry, New York, N.Y. 10021 Although the biochemical and biophysical properties of collagen are well known, its surface properties are poorly understood. Collagen-containing structures, which are exposed to blood when endothelium is injured, initiate clot formation. Surface properties of restructured collagen have important biomedical applications. Several types of collagen films with varying surface morphologies were prepared, crosslinked with aldehydes, and implanted in rabbits. Results indicate that biologic degradation of collagen can be controlled and delayed for at least 90 days. Inflammatory reactions are minimal. Crosslinking decreases swelling ratios and increases shrinkage temperature and resistance to bacterial collagenase. These studies are a base to develop collagen for specific biomaterials and to study collagen surface-blood interactions. Collagen ^

has e v o l v e d i n n a t u r e as the p r i m a r y c o n n e c t i v e tissue p r o t e i n

i n a n i m a l s . E l e c t r o n m i c r o g r a p h s of c o l l a g e n f r o m w i d e l y d i v e r g e n t

species r e v e a l f e w , i f a n y , differences.

A s a s u p p o r t i n g structure a n d as a

surface for g r o w t h of cells, c o l l a g e n makes a n a t t r a c t i v e b i o m a t e r i a l . M e t h o d s exist for s o l u b i l i z i n g large amounts of c o l l a g e n a n d for r e s t r u c t u r i n g it i n t o a v a r i e t y of forms for b i o m e d i c a l a p p l i c a t i o n s ( I , 2 ) . Surface p r o p e r t i e s of this u b i q u i t o u s p r o t e i n , w h i c h are of p a r a m o u n t i m p o r t a n c e for m a n y of these a p p l i c a t i o n s , are, h o w e v e r , the least s t u d i e d a n d least u n d e r s t o o d . T h e s t r u c t u r e a n d p h y s i c a l properties of c o l l a g e n are w e l l k n o w n . S e v e r a l recent r e v i e w s are a v a i l a b l e to i n t e r e s t e d readers (3, 4,

5,6,7,8).

S o m e of the w o r k p e r t i n e n t to the p r o b l e m s of u s i n g c o l l a g e n as a b i o m a t e r i a l a l o n g w i t h some of o u r recent w o r k o n r e c o n s t i t u t i n g c o l l a g e n surfaces are r e v i e w e d

here. 26

Baier; Applied Chemistry at Protein Interfaces Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

2.

STENZEL E T AL.

Collagen

27

Surfaces

C o l l a g e n m o l e c u l e s , t r o p o c o l l a g e n , consist of three p e p t i d e chains w o u n d together as a t r i p l e h e l i x . T h e m o l e c u l e is m a r k e d l y a s y m m e t r i c a l w i t h a l e n g t h of 2800 A a n d a w i d t h of 15 A . It r e a d i l y p o l y m e r i z e s to form

fibrils

a n d fibers, a n d i n n a t u r e i t exists l a r g e l y as a n i n s o l u b l e

m a c r o m o l e c u l a r c o m p l e x of c r o s s l i n k e d m o l e c u l e s .

O n l y small amounts

of n a t i v e c o l l a g e n are s o l u b l e , either i n d i l u t e a c i d or salt solutions. M o s t of the c h e m i c a l a n d p h y s i c a l studies o n the s t r u c t u r e of c o l l a g e n h a v e been done w i t h acid-soluble preparations. O f prime importance i n using r e c o n s t i t u t e d c o l l a g e n as a b i o m a t e r i a l w a s the d i s c o v e r y of n o n - h e l i c a l appendages, t e r m e d telopeptides, o n t h e b a s i c t r o p o c o l l a g e n t r i p l e h e l i x .


F . O . Schmitt's g r o u p at M I T f o u n d that p r o t e o l y t i c e n z y m e s , s u c h as p e p s i n a n d t r y p s i n , d i g e s t e d a s m a l l p o r t i o n of t r o p o c o l l a g e n b u t left the t r i p l e h e l i x i n t a c t ( 9 ) .

T r e a t m e n t of t r o p o c o l l a g e n w i t h p r o t e o l y t i c

e n z y m e s , other t h a n collagenase,

a l t e r e d the i n t e r a c t i o n properties

c o l l a g e n b u t d i d not result i n d e n a t u r a t i o n or d e g r a d a t i o n .

of

Nishihara

a n d M i y a t a ( 1 ) d e v e l o p e d t e c h n i q u e s for s o l u b i l i z i n g a n d p u r i f y i n g l a r g e q u a n t i t i e s of i n s o l u b l e c o l l a g e n w i t h c o n t r o l l e d proteolysis at a l o w p H . T h e s o l u b i l i z e d c o l l a g e n is easily p u r i f i e d b y r e p e a t e d p r e c i p i t a t i o n , w a s h ing, a n d resolubilization. B o t h physical a n d chemical methods have been u s e d for r e s t r u c t u r i n g or r e c r o s s l i n k i n g the e n z y m e - s o l u b i l i z e d c o l l a g e n as specific

biomaterials.

Virtually

a l l of

our work

has u t i l i z e d this

enzyme-solubilized collagen material. T h e i n d i v i d u a l p e p t i d e c h a i n s assume a r a n d o m c o i l c o n f i g u r a t i o n w h e n c o l l a g e n is d e n a t u r e d a n d c a n exist i n a n y one of three states. I f the three p o l y p e p t i d e strands are not c o v a l e n t l y l i n k e d , the c o l l a g e n is k n o w n as a l p h a c o l l a g e n .

If t w o of the strands are c o v a l e n t l y c r o s s l i n k e d , the

c o l l a g e n is k n o w n as b e t a c o l l a g e n . A l l three p o l y p e p t i d e chains l i n k e d together is k n o w n as g a m m a c o l l a g e n . T h e i n d i v i d u a l chains themselves c a n exist i n either of t w o species, e a c h w i t h a s i m i l a r b u t d i s t i n c t p r i m a r y structure. T h e t w o most c o m m o n types of c o l l a g e n p o l y p e p t i d e c h a i n s f o u n d i n m a m m a l i a n tissues are k n o w n as a l p h a - 1 a n d a l p h a - 2 chains. M o s t collagens are m a d e u p of t w o a l p h a - 1 a n d one a l p h a - 2 chains, a l t h o u g h there are significant differences i n this m a k e u p w h i c h w i l l be n o t e d later. B o t h of the chains h a v e a f a i r l y t y p i c a l sequence of a m i n o acids c h a r a c t e r i z e d b y a r e p e a t i n g u n i t o f three a m i n o acids, g l y c i n e a p p e a r i n g at e v e r y t h i r d p o s i t i o n .

T h e r e are l a r g e n u m b e r s of i m i n o

acids, e s p e c i a l l y p r o l i n e a n d h y d r o x y p r o l i n e .

These probably

account

for the t y p i c a l t e r t i a r y s t r u c t u r e of the m o l e c u l e . T h e p o l y p e p t i d e c h a i n s c o n t a i n a b o u t 40 sets of p o l a r a n d a p o l a r a m i n o a c i d groups.

These

groups m a y be e x t r e m e l y i m p o r t a n t i n the surface properties of c o l l a g e n materials.

T h e t y p i c a l r e p e a t i n g sequence w i t h g l y c i n e i n the

third

p o s i t i o n does not exist for the t e l o p e p t i d e e n d regions. T h i s p a r t of t h e m o l e c u l e is not i n a h e l i c a l c o n f i g u r a t i o n .


28

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

C o l l a g e n contains c a r b o h y d r a t e , p r e d o m i n a n t l y i n the f o r m of g l y c o s y l a t e d h y d r o x y l y s i n e residues, as 0 - g a l a c t o s y l - / ? - g l u c o s y l side c h a i n s . T h e i n t e r m o l e c u l a r crosslinks i n c o l l a g e n o c c u r w h e n t h e e n z y m a t i c o x i d a t i o n of l y s i n e residues b y l y s i n e oxidase spontaneously c r o s s l i n k via aldimine a n d aldol bonds ( J O ) . I m m u n o l o g i c properties m u s t b e c o n s i d e r e d w h e n a n y p r o t e i n m a t e r i a l is to b e u s e d as a n i m p l a n t or surface for b l o o d flow i n a f o r e i g n species.

S i n c e c o l l a g e n has c h a n g e d v e r y little i n the course of e v o l u

t i o n , there are f e w a n t i g e n i c d e t e r m i n a n t s that are r e c o g n i z e d i n h e t e r ologous systems.

T h e strongest of these d e t e r m i n a n t s appears to reside


i n t h e protease l a b i l e , n o n - h e l i c a l e n d regions.

T h e s e are k n o w n as

P-specific antigens a n d are f o u n d i n the 300 A r e g i o n at t h e C t e r m i n u s of b o t h types o f a l p h a c h a i n s . T h e s e are species specific a n d protease labile.

Α-specific antigens h a v e a g e n e r a l c o l l a g e n specificity, a n d they

crossreact a m o n g species a n d w i t h e n z y m e - t r e a t e d collagen.

S-specific

d e t e r m i n a n t s are species specific a n d crossreact w i t h b o t h n a t i v e a n d e n z y m e - t r e a t e d c o l l a g e n ( J J , 12).

E n z y m e - s o l u b i l i z e d c o l l a g e n , there

fore, lacks one of the strongest a n t i g e n i c d e t e r m i n a n t s of the m o l e c u l e . C r o s s l i n k i n g w i t h a v a r i e t y of reagents d i m i n i s h e s the i m m u n o l o g i c ac t i v i t y e v e n f u r t h e r . F r o m a p r a c t i c a l or c l i n i c a l p o i n t of v i e w , a n t i g e n i c i t y has not b e e n a p r o b l e m e v e n w h e n m a t e r i a l s s u c h as t r e a t e d c a r o t i d arteries h a v e b e e n p l a c e d i n f o r e i g n species.

bovine

T h u s , a l t h o u g h the

a n t i g e n i c structure of c o l l a g e n is i m p o r t a n t i n u n d e r s t a n d i n g its b i o c h e m istry, i t is of m i n o r i m p o r t a n c e i n its c l i n i c a l a p p l i c a t i o n . M o r e p e r t i n e n t to the p r o b l e m of surface s t r u c t u r e is the effect of c o l l a g e n o n v a r i o u s c l o t t i n g factors.

W h e n t h e e n d o t h e l i u m of

blood

vessels is i n j u r e d or d a m a g e d , platelets a d h e r e to the exposed s u b e n d o t h e l i a l m a t e r i a l , a n d a c o m p l e x set of reactions t h a t l e a d to t h r o m b o s i s a n d hemostasis is i n i t i a t e d (13).

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

a n d other c o n n e c t i v e tissue proteins, b u t the c o l l a g e n , e s p e c i a l l y , has b e e n i m p l i c a t e d as a n i n i t i a t o r of the c l o t t i n g m e c h a n i s m . W h e n c o l l a g e n is a d d e d to p l a t e l e t - r i c h p l a s m a , the platelets a g g l u t i n a t e (14).

Collagen

has also b e e n s h o w n to activate H a g e m a n factor, or F a c t o r X I I

(15).

N u m e r o u s studies h a v e i n d i c a t e d that the cluster of c h a r g e d groups a l o n g the c o l l a g e n fibrils are i m p o r t a n t i n these reactions (14, 15, 16).

More

i n t e r e s t i n g is w h a t takes p l a c e at the c o l l a g e n surface, b u t f e w studies d i r e c t themselves to this p r o b l e m . R e c e n t l y , Jaffe a n d D e y k i n ( J 6 ) p r e s e n t e d e v i d e n c e for a s t r u c t u r a l r e q u i r e m e n t for c o l l a g e n - i n d u c e d p l a t e l e t a g g r e g a t i o n .

T h e y found that

p a r t i c u l a t e , s a l t - p r e c i p i t a t e d c o l l a g e n was i n a c t i v e i n terms of p l a t e l e t agglutination a n d that soluble monomeric collagen resulted i n platelet a g g l u t i n a t i o n o n l y after a l a g of a b o u t 3 m i n .

Soluble microfibrillar


2.

STENZEL E T A L .

29

Collagen Surfaces

c o l l a g e n , h o w e v e r , w a s as a c t i v e as n a t i v e p a r t i c u l a t e c o l l a g e n i n aggreg a t i n g platelets. T h i s e v i d e n c e suggests that t r o p o c o l l a g e n is not sufficient for p l a t e l e t a g g r e g a t i o n n o r is r a n d o m l y p r e c i p i t a t e d ( s a l t ) c o l l a g e n , b u t that a n a r c h i t e c t u r a l a r r a n g e m e n t of the molecules is r e q u i r e d to i n i t i a t e p l a t e l e t aggregation.

T h e s e r e q u i r e m e n t s m a y also be necessary

for p l a t e l e t a d h e s i o n . C o l l a g e n i n basement m e m b r a n e has a different q u a r t e r n a r y structure f r o m s k i n or fibrous c o l l a g e n (17,18).

T h i s t y p e of c o l l a g e n consists

of three a-l c h a i n s , has a h i g h g l y c o s y l a t e d h y d r o x y l y s i n e content, a n d a p p a r e n t l y has d i s u l f i d e b o n d s c o n n e c t i n g i t to other p r o t e i n constituents.


T h e s e d i s u l f i d e b o n d s m a y be l o c a t e d i n the t e l o p e p t i d e , or n o n - h e l i c a l , r e g i o n of t h e m o l e c u l e . T h e surface of basement m e m b r a n e m a y p r o v i d e a c l u e to the p h y s i o l o g i c a l a r c h i t e c t u r e r e q u i r e d for i n i t i a t i o n of t h r o m bosis. S u c h k n o w l e d g e w o u l d be of use, not o n l y i n b i o m a t e r i a l s research, b u t also i n u n d e r s t a n d i n g v a r i o u s k i d n e y diseases a n d p o s s i b l y

even

atherosclerosis. S e v e r a l n a t u r a l c o l l a g e n m a t e r i a l s are c u r r e n t l y b e i n g u s e d i n c l i n i c a l m e d i c i n e . B o v i n e c a r o t i d heterografts, for instance, f u n c t i o n w e l l i n m a n (19).

T h e s e grafts are p r e p a r e d b y c l e a n i n g b o v i n e

c a r o t i d arteries,

treating them w i t h a proteolytic enzyme (ficin), and crosslinking them w i t h a n a l d e h y d e ( d i a l d e h y d e starch ). A u t o l o g o u s a n d h o m o l o g o u s v e i n grafts are also u s e d c l i n i c a l l y .

T h e e n d o t h e l i u m varies f r o m r e l a t i v e l y

p o o r l y p r e s e r v e d to v i r t u a l l y absent i n these v e i n grafts. T h e b o v i n e vessels are a l l p l a c e d i n areas of r e l a t i v e l y h i g h flow a n d , i n the case of v e i n grafts, e n d o t h e l i a l i z a t i o n occurs over the surfaces

(20).

T h e c r o s s l i n k i n g reagent is i m p o r t a n t i n the case of b o v i n e c a r o t i d heterografts.

C h r o m e - t a n n e d grafts h a d a h i g h i n c i d e n c e of t h r o m b o s i s

whereas a l d e h y d e - t r e a t e d ones f u n c t i o n e d w e l l .

F o r m a l i n - t r e a t e d grafts

t e n d e d to b e w e a k , b u t g l u t a r a l d e h y d e - t r e a t e d ones d i d not r u p t u r e A v a r i e t y of m e t h o d s

for p r e p a r i n g c o l l a g e n

films

from

(21).

enzyme-

s o l u b i l i z e d , m o n o m e r i c c o l l a g e n w i t h different surface structures w e r e evaluated.

O u r i n i t i a l interest w a s to d e t e r m i n e the effects of v a r i o u s

p r e p a r a t i v e t e c h n i q u e s o n the in vivo b e h a v i o r of the

films.

Experimental I n m e t h o d I, the c o l l a g e n s o l u t i o n w a s p o u r e d onto a m e t h y l m e t h a crylate plate a n d lowered into a 0.02M dibasic N a H P 0 solution ( p H 8.5) c o n t a i n i n g 0 . 1 % g l u t a r a l d e h y d e . C o l l a g e n p r e c i p i t a t e s as n a t i v e t y p e fibrils i n p h o s p h a t e buffer at this p H . T h e films w e r e c r o s s l i n k e d as the fibers w e r e f o r m i n g before the films w e r e d r i e d . C r o s s l i n k i n g w a s a l l o w e d to c o n t i n u e for 7 or 24 hrs. T h e films w e r e w a s h e d w i t h w a t e r , plasticized w i t h 2 % glycerine, and air dried. I n m e t h o d I I , the c o l l a g e n w a s not p r e c i p i t a t e d . A c i d i c solutions of c o l l a g e n w e r e a i r d r i e d a n d the r e s u l t i n g films w e r e c r o s s l i n k e d u s i n g 2

4


30

APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

0 . 5 % g l u t a r a l d e h y d e i n . 0 2 M N a H P 0 for 10 or 20 m i n s . T h e s e films w e r e also w a s h e d w i t h w a t e r a n d p l a s t i c i z e d . I n m e t h o d I I I , the c o l l a g e n w a s first p r e c i p i t a t e d i n p h o s p h a t e buffer, as i n m e t h o d I , b u t the film w a s d r i e d p r i o r to c r o s s l i n k i n g . T h e crossl i n k i n g w a s p e r f o r m e d as i n m e t h o d I I . A n o t h e r series of films w a s p r e p a r e d b y the same m e t h o d s b u t crosslinked w i t h dialdehyde starch rather than w i t h glutaraldehyde. T h e c r o s s l i n k i n g c o n d i t i o n s w e r e s i m i l a r to those u s e d f o r t h e g l u t a r a l d e h y d e c r o s s l i n k e d films w i t h the e x c e p t i o n of m e t h o d I I I . T h e s e latter films w e r e p r e p a r e d b y m i x i n g c o l l a g e n a n d d i a l d e h y d e starch at a r a t i o of 1 to 1.5. T h i s m i x t u r e w a s t h e n d i a l y z e d against . 0 2 M N a H P 0 to p r e c i p i t a t e c o l l a g e n fibers. A f t e r c r o s s l i n k i n g , the films w e r e w a s h e d , p l a s ticized, and dried. C o n t r o l films of e a c h t y p e w e r e also p r e p a r e d u s i n g t h e same p r o c e d u r e s b u t w i t h o u t the c r o s s l i n k i n g reagent. A t h i r d g r o u p of films w a s p r e p a r e d , u s i n g a m i x t u r e of 2 0 % e n z y m e s o l u b i l i z e d c o l l a g e n a n d 8 0 % i n s o l u b l e c o l l a g e n to increase the i n i t i a l film strength. T h e s e films w e r e c r o s s l i n k e d u s i n g g l u t a r a l d e h y d e i n the same m a n n e r as the e n z y m e - s o l u b i l i z e d c o l l a g e n films. S i n c e the s o l u tions c o n t a i n e d a h i g h i n i t i a l content of fiber, m e t h o d I I I w a s e l i m i n a t e d i n this g r o u p . C o n t r o l films of e a c h w e r e also m a d e b y e l i m i n a t i n g the glutaraldehyde. 2

4


2

Table I.

4

Swelling Ratios of Collagen Films

Glutaraldehyde

Dialdehyde

Starch

Method

Control

Crosslinked

Control

Crosslinked

I II III

19 12 19

2 2 2

19 12 19

2 2 2

Composite 80% Insoluble Collagen Fiber—20% Soluble Collagen Crosslinked

Control

2 2

Results S w e l l i n g ratios, s h r i n k a g e t e m p e r a t u r e s , a n d resistance to collagenase w e r e m e a s u r e d for a l l the

films.

S w e l l i n g ratios w e r e d e t e r m i n e d

m e a s u r i n g t h e c h a n g e i n w e i g h t before a n d after h y d r a t i o n of the

by

films.

C r o s s l i n k i n g decreased the s w e l l i n g ratios as c o m p a r e d w i t h c o n t r o l films (Table

I).

S h r i n k a g e temperatures w e r e d e t e r m i n e d as a n i n d i c a t o r of the den a t u r a t i o n t e m p e r a t u r e of t h e

films.

I n a l l cases, c r o s s l i n k i n g g r e a t l y

increased shrinkage temperatures for each preparation. T h u s , crosslinking s t a b i l i z e s c o l l a g e n m o l e c u l e s a n d retards d e n a t u r a t i o n ( T a b l e I I ) . A n o t h e r i m p o r t a n t aspect of these films is t h e i r resistance to d e g r a d a tion by biologic

substances

l i k e collagenase.

E a c h of the

films


was

CoUagen

STENZEL ET A L .

Table I I .

Shrinkage Temperatures

Dialdehyde Starch Collagen Films

Glutaraldehyde


31

Surfaces

Method

Control

Crosslinked

Control

Crosslinked

I II III

45.0°C 45.8 45.0

66.0°C 71.8 78.1

45.0°C 45.8 45.0

65.0°C 61.5 67.0

Composite 80% Insoluble Collagen Fiber—20% Soluble Collagen Control

Crosslinked

47.5°C 48.0

77.0°C 71.0

i n c u b a t e d for 2 hrs at 37 ° C w i t h b a c t e r i a l collagenase.

This

enzyme

is less specific a n d m o r e d e s t r u c t i v e t h a n m a m m a l i a n collagenase.

The

d e g r a d a t i o n of the films was m e a s u r e d b y the m i c r o m o l e s of a m i n o acids released p e r m i l l i g r a m

film.

I n each case, the c r o s s l i n k i n g i n c r e a s e d r e

sistance to b a c t e r i a l collagenase. I n some cases the films w e r e c o m p l e t e l y resistant to it ( T a b l e I I I ) . E a c h t y p e of

film

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

m u s c u l a r l y i n r a b b i t s to assess tissue r e a c t i o n a n d rate of r e s o r p t i o n . T h e film w a s c a r e f u l l y cut i n t o a c i r c l e w i t h a d i a m e t e r of 10 m m . S h a m incisions w e r e also m a d e i n each r a b b i t as controls for the a m o u n t of i n f l a m m a t i o n r e s u l t i n g f r o m s u r g i c a l t r a u m a alone. k i l l e d after 7 to 180 days.

T h e animals were

T h e i m p l a n t s w e r e e x a m i n e d grossly for

i n f l a m m a t i o n , changes i n size, a n d a p p e a r a n c e of the

films.

Histologic

p r e p a r a t i o n s w e r e also m a d e of e a c h film to e x a m i n e the c e l l u l a r r e a c t i o n . B y 7 days, i n f l a m m a t o r y cuffs h a d e n c i r c l e d the i m p l a n t s . T h e c o n t r o l films

s h o w e d e v i d e n c e of b e i n g d i g e s t e d ; t h e y w e r e s w o l l e n , w e a k e r ,

and usually thinner. F i g u r e 1 is a c o n t r o l ( n o t c r o s s l i n k e d ) film p r e p a r e d b y m e t h o d I I w i t h enzyme-solubilized collagen.

Table I I I .

Resistance to Collagenase"

Glutaraldehyde

α

T h e film w a s r e m o v e d after 7 days

Dialdehyde

Starch

Method

Control

Crosslinked

Control

Crosslinked

I II III

0.230 0.069 0.218

0.028 0.025 0.000

0.230 0.069 0.218

0.000 0.005 0.004

Composite 80%) Insoluble Collagen Fiber—20% Soluble Collagen Control

Crosslinked

0.473 0.159

0.000 0.000

—

In micromoles of amino acids released per milligram of sample.


—


32

Figure

APPLIED

CHEMISTRY

AT PROTEIN

INTERFACES

1.

Control (not crosslinked) collagen film removed after 7 days subcutaneous implantation in rabbits. XI28

Figure 2.

Control (not crosslinked) collagen film removed after 14 days intramuscular implantation in rabbits. XI28



2.

STËNZEL E T AL.

Figure

of

3.

Collagen

33

Surfaces

Method II glutaraldehyde crosslinked collagen film removed after 90 days subcutaneous implantation in rabbits. XI28

subcutaneous

implantation.

N o t e the f o r e i g n b o d y t y p e

of

tissue

r e a c t i o n w i t h histiocytes a n d b e g i n n i n g r e s o r p t i o n of the c o l l a g e n . F i g u r e 2 is the same t y p e of film r e m o v e d

after 14 days of i n t r a -

m u s c u l a r i m p l a n t a t i o n . H e r e the c o l l a g e n is f r a g m e n t e d a n d i n f i l t r a t e d w i t h i n f l a m m a t o r y cells.

A f t e r 21 days, a l l c o n t r o l

films

disappeared

except for the ones p r e p a r e d f r o m a m i x t u r e of i n s o l u b l e c o l l a g e n a n d enzyme-solubilized collagen. The

i n f l a m m a t o r y response w i t h

crosslinked

a n d not d i s s i m i l a r to the s h a m operations. flammation

at d a y 7.

fiber

S o m e of these r e m a i n e d for 30 days. films

was

minimum

G r o s s l y , there w a s l i t t l e i n -

W h a t inflammatory reaction appeared,

peaked

a r o u n d d a y 14. T h i s w a s c h a r a c t e r i z e d b y a n e n l a r g e d cuff of cells surr o u n d i n g the i m p l a n t o c c a s i o n a l l y a c c o m p a n i e d b y a

fluid

exudate.

A l l of the c r o s s l i n k e d i m p l a n t s l a s t e d for at least 60 days. t i m e , a f e w of the e n z y m e - s o l u b i l i z e d

collagen

films

A t this

crosslinked

with

either g l u t a r a l d e h y d e or d i a l d e h y d e starch a p p e a r e d to be t h i n n e r . S o m e of the films c r o s s l i n k e d w i t h g l u t a r a l d e h y d e a p p e a r e d to be

somewhat

t h i n n e r after 90 days, a l t h o u g h most w e r e intact. F i g u r e 3 is a m e t h o d I I e n z y m e - s o l u b i l i z e d c o l l a g e n film c r o s s l i n k e d w i t h g l u t a r a l d e h y d e a n d i m p l a n t e d subcutaneously.

It was

unchanged

after 90 days.


34

APPLIED CHEMISTRY

AT PROTEIN

T h e films p r e p a r e d f r o m a m i x t u r e of c o l l a g e n

fiber

INTERFACES

a n d soluble

c o l l a g e n c r o s s l i n k e d w i t h g l u t a r a l d e h y d e w e r e a l l i n t a c t after 90 days implantation. F i g u r e 4 is a m e t h o d I I c u l a r l y for 90 days.

fiber

film

that w a s i m p l a n t e d i n t r a m u s -

T h e t h i n , d e l i c a t e s u r r o u n d i n g fibrous tissue c a n

b e seen p a r t i a l l y a d h e r i n g to the surface of t h e

film.

T h e 180-day

films

h a v e not yet b e e n r e m o v e d . Discussion T h e s e results i n d i c a t e t h a t the b i o l o g i c d e g r a d a t i o n of c o l l a g e n c a n Downloaded by CORNELL UNIV on September 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch002

b e c o n t r o l l e d a n d d e l a y e d f o r at least 90 days. It is r e l a t i v e l y u n i m p o r tant w h e t h e r c o l l a g e n is c r o s s l i n k e d i n the w e t or the d r y state or w h e t h e r the films are p r e c i p i t a t e d or not p r i o r to c r o s s l i n k i n g . B o t h g l u t a r a l d e h y d e a n d d i a l d e h y d e s t a r c h are effective i n s t a b i l i z i n g c o l l a g e n m o l e c u l e s a n d r e t a r d i n g t h e i r b i o - d e g r a d a t i o n for at least 90 days a n d p e r h a p s longer. T h e i n f l a m m a t o r y response has b e e n m i n i m u m , r e a c h i n g a p e a k at 14 days a n d t h e n s u b s i d i n g . T h e films are e v e n t u a l l y c o v e r e d b y a t h i n , m e m b r a n e w h i c h i n some cases adheres to the surface of the

fibrous

film.

Figure 4. Method II glutaraldehyde crosslinked composite collagen fibersoluble collagen film removed after 90 days intramuscular implantation in rabbits. XI28


2.

STENZEL E T AL.

Collagen

Surfaces

35

T h e s e studies p r o v i d e a base f o r f u r t h e r d e v e l o p m e n t o f c o l l a g e n materials f o r specific b i o m a t e r i a l a p p l i c a t i o n s . F u r t h e r studies are b e i n g d i r e c t e d to t h e effect o f c o l l a g e n film o n b l o o d i n terms of platelet a n d white cell adhesion, protein absorption, a n d thrombogenicity.


References 1. Nishihara, T., Miyata, T., Collagen Symp. Jap. (1962) 3, 66-84. 2. Drake, M. P., Davidson, P. F., Bump, S., Schmitt, F. O., Biochemistry (1966) 5, 301-312. 3. Gallop, P. M., Blumenfeld, O. O., Seifter, S., Ann. Rev. Biochem. (1972) 41, 617-672. 4. Bailey, A. J., "Comprehensive Biochemistry," M. Florkin and Ε. H. Stotz, Eds., 26, pp. 297-423, American Elsevier, New York, 1968. 5. Traub, W., Piez, Κ. Α., Advan. Prot. Chem. (1971) 25, 243-352. 6. Piez, Κ. Α., Ann. Rev. Biochem. (1968) 37, 547-571. 7. Yannas, I. V., J. Macromol. Sci. Rev. Macromol. Chem. (1972) C7 (1), 49-104. 8. Stenzel, Κ. H., Miyata, T., Rubin, A. L., Ann. Rev. Biophys. Bioeng. (1974) 3, 231-253. 9. Rubin, A. L., Pfahl, D., Speakman, P. T., Davidson, P. F., Schmitt, F. O., Science (1973) 13, 37-38. 10. Tanzer, M. L., Science (1973) 180, 561-566. 11. Steffen, C., Timpl, R., Wolff, I., Immunology (1968) 15, 135-144. 12. Timpl, R., Wolff, I., Wick, G., Furthmayr, H., Steffen, C., J. Immunol. (1968) 101, 725-729. 13. Baumgartner, H. R., Handenschild, C., Ann. N.Y. Acad. Sci. (1972) 201, 22-36. 14. Wilner, G. D., Nossel, H. L., LeRoy, E. C., J. Clin. Invest. (1968) 47, 2616-2621. 15. Ibid. (1968) 47, 2608-2615. 16. Jaffe, R., Deykin, D., J. Clin. Invest. (1974) 53, 875-883. 17. Kefalides, Ν. Α., Biochem. Biophys. Res. Commun. (1971) 45, 226-231. 18. Kefalides, Ν. Α., Int. Rev. Conn. Tiss. Res. (1973) 6, 63-104. 19. Rosenberg, N., Lord, G. H., Henderson, J., Bothwell, J. W., Gaughran, E. R. L., Surgery (1970) 65, 951-956. 20. Reichle, R. Α., Stewart, G. J., Essa, H., Surgery (1973) 74, 945-960. 21. Bothwell, J. W., Lord, G. H., Rosenberg, N, Burrowes, C. B., Wesolowski, S. Α., Sawyer, P. N., "Biophysical Mechanisms in Vascular Homeostasis and Intravascular Thrombosis," P. N. Sawyer, Ed., pp. 306-313, Appleton-Century-Crofts, New York, 1965. RECEIVED July 7, 1974. Work was supported in part by the National Science Foundation and John A. Hartford Foundation.


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