Upsala J Med Sci 88: 25-31, 1983 Composition of Surface Layers in Unfixed Autologous Fascia Lata Heart Valve Grafts A transmission electron microscopical study Roger WillCn’, Helena WillCn’, Wieslaw T. Dubie12 and Lennart Johansson’ From the Department of Histopathology’. University Hospital, Lund, and Department of Thoracic and Cardiovascular Surgery’, University Hospital, Uppsala, Sweden ABSTRACT Sixty-nine p a t i e n t s underwent a o r t i c valve replacement w i t h frame mounted non-fixated f a s c i a l a t a g r a f t s . Nine p a t i e n t s s t i l l have t h e i r f a s c i a l a t a valve i n p l a c e a f t e r a period of between 9 t o 1 1 y e a r s . Fifty-two g r a f t s were a v a i l a b l e f o r h i s t o - p a t h o l o g i c examination. The mechanism of valve t h i c k e n i n g was s t u d i e d by t r a n s m i s s i o n e l e c t r o n microscopy ( T E M ) . The major f a c t o r c o n t r i b u t i o n t o t h e t h i c k e n i n g seems t o be a s u r f a c e accumulation of complex p r o t e i n s , f i b r i n and p l a t e l e t s . W i t h i n t h e v a l v u l a r t i s s u e i t s e l f , s u b - c e l l u l a r d e g e n e r a t i v e products i n c e l l cytoplasm, t h e i n t e r s t i t i u m and i n t h e sub-surface l a y e r a l s o c o n t r i b u t e d . The t h i c k e n i n g never exceeded one f i f t h of t h e o r i g i n a l t h i c k n e s s of t h e valve. INTRODUCTION Unfixed autologous f a s c i a l a t a h e a r t valve g r a f t m a t e r i a l i s f a i r l y rough on one s u r f a c e , with s l i g h t l y p r o t r u d i n g smooth c o l l a g e n bundles a t t h e o t h e r ( 1 4 ) . The rough s i d e i s i n i t i a l l y covered by f a t , c o n n e c t i v e t i s s u e and v e s s e l s . A f t e r s u r g i c a l t a i l o r i n g , some remnants of small damaged f i b e r s of f a s c i a l t i s s u e remain. Implanted i n t h e blood s t r e a m , t h e f a s c i a l tissue i s r a p i d l y covered by a f i n e , g r a n u l a r m a t e r i a l , probably p r o t e i n ( 2 , 1 4 ) . Small fragments of f i b r i n and p l a t e l e t s can be v i s u a l i z e d (14). T h i s covering makes t h e s u r f a c e smoother and may e x p l a i n t h e f r e q u e n t l a c k of c l i n i c a l thrombus- formation w i t h t h e s e p r o s t h e s e s ( 6 , 10, 1 1 ) . The blood born elements which adhere t o t h e s u r f a c e l a t e r become organized mainly on t h e rough s u r f a c e . Eventually t h e v a l v u l a r t i s s u e i t s e l f undergoes f o c a l d e g e n e r a t i v e and hyper- p l a s t i c changes ( 3 , 4 , 151, probably a s a r e s u l t of mechanical s t r e s s ( 5 ) . D i f f e r e n t s t a g e s o f t i s s u e breakdown can be i l l u s t r a t e d ( 1 4 , 1 5 ) . F i n a l l y , shrinkage c a l c i f i c a t i o n and v a l v e t h i c k e n i n g appear i n some of t h e valves w i t h subsequent f a i l u r e due t o r u p t u r e o r dysfunction (4). The aim of t h i s paper i s t o d i s c u s s t h e p o s s i b l e c o n t r i b u t i o n t o valve t h i c k e n i n g by the endogenous breakdown of f a s c i a l tissue i t s e l f which becomes enveloped by the 25 s u r f a c e pseudomembrane. PATIENTS Between November 13, 1969, and June 7 , 1972, 69 p a t i e n t s underwent i s o l a t e d a o r t i c valve replacement w i t h a frame-mounted f a s c i a l a t a g r a f t . Of the 55 p a t i e n t s r e l e a s e d from t h e h o s p i t a l , 30 d i e d a f t e r between 5 months and 83 y e a r s . In 3 o f t h e s e c a s e s t h e cause of d e a t h was g r a f t f a i l u r e and, i n 2 7 , myocardial f a i l u r e sometimes i n combination w i t h s e p t i c i n f e c t i o n ( 5 p a t i e n t s ) . Twenty-two p a t i e n t s were r e o p e r a t e d due t o g r a f t f a i l u r e w i t h a o r t i c r e g u r g i t a t i o n and 6 of t h e s e died during o r soon a f t e r t h e r e o p e r a t i o n . Nine p a t i e n t s s t i l l have t h e i r o r i g i n a l f a s c i a l a t a valve g r a f t i n p l a c e a f t e r a period of between 9 and 1 1 y e a r s and most of them a r e doing well ( f i g . 1 ) . Died q x o r postop Died in cardiae failure, 5 with septic infection. ,6 op. or postop. dead \16 well with other 22 Reop. prosthesis. 9 Well with fascialata graft. F i g . 1 . C l i n i c a l m a t e r i a l . Ultimate f a t e of 69 f a s c i a l a t a h e a r t a o r t i c valve o p e r a t e d p a t i e n t s . METHODS The t i s s u e was inbedded f o r scanning e l e c t r o n microscopy (SEM) a s p r e v i o u s l y d e s c r i b e d ( 1 4 , 1 5 ) . In o r d e r t o c a r r y o u t t r a n s m i s s i o n e l e c t r o n microscopy (TEM) t h e scanning e l e c t r o n microscopy t i s s u e was trimmed i n t o small blocks 3 x 3 mm and p u t i n t o a mixture of propylenoxide/epon 1 : l over n i g h t . The following morning t h e t i s s u e was l e f t i n epon f o r 4 t o 6 hours. I t was then p u t i n BeemR-capsules f i l l e d w i t h f r e s h l y made r e s i n mixture and hardened a t 6OoC f o r 3 t o 4 days. The blocks were c u t i n t h e usual way on a 26 L K B u l t r a t o m e R . The s e c t i o n s were mounted on formvar c o a t e d grids, s t a i n e d w i t h l e a d - c i t r a t e f o r 4 minutes, rinsed and d r i e d , c o u n t e r s t a i n e d w i t h uranyl- a c e t a t e f o r 30 minutes a t 37OC, and f i n a l l y r i n s e d and d r i e d . The gold l a y e r p u t on t h e v a l v u l a r s u r f a c e i n the p r e p a r a t i o n f o r t h e SEM i n v e s t i g a t i o n served a s a marker f o r i d e n t i f i c a t i o n of f a s c i a 1 s u r f a c e . RESULTS Central p a r t s of t h e normal v a l v u l a r t i s s u e showed a r e a s w i t h well pre- served f i b r o c y t e s and f i brobl a s t s surrounded by numerous col 1 agen f i b e r s ( 1 4 , 1 5 ) . Sometimes markedly degenerated a r e a s were observed, showing f i b r o b l a s t s with e n d o c y t i c a c t i v i t y , and i n t e r s t i t i a l t i s s u e which c o n t a i n e d amorphous m a t e r i a l and only a very few c o l l a g e n f i b r i l s ( f i g . 2 ) . Some of t h e c e l l s contained p a r t l y degenerated rough and smooth endopl asmic r e t i c u l u m , primary and secondary lysosomes and o t h e r c e l l d e b r i s ( f i g s . 3 , 4 ) . Some f i b r o b l a s t s Fig. 2 . E l e c t r o n micrograph of f a i r l y well preserved f i b r o b l a s t with endo- c y t i c a c t i v i t y (arrow) and s l e n d e r p r o t r u s i o n s . Degeneration i s e v i d e n t i n t h e i n t e r c e l l u l a r space where mostly amorphous material and only s c a n t c o l l a g e n bundles a r e seen ( o r i g . mag. x 5 0 0 0 ) . showed s l i g h t bulging of t h e plasma membrane i n a s s o c i a t i o n w i t h lysosomes and o u t s i d e t h e c e l l e x p e l l e d r e s i d u a l bodies were v i s u a l i z e d ( f i g . 4 ) . Sometimes t h e i n t e r s t i t i u m was f i l l e d w i t h well preserved c o l l a g e n bundles showing c r o s s s t r i a t i o n ( f i g . 5 ) . S t u d i e s of a r e a s c l o s e r t o t h e s u r f a c e showed abundant c o l l a g e n f i b r i l s t o g e t h e r w i t h l i g h t ground s u b s t a n c e , c e l l d e b r i s and degenerated p l a t e l e t s ( f i g . 6 ) . I t was thus p o s s i b l e t o f i n d s u b - c e l l u l a r breakdown products w i t h i n a l l p a r t s of t h e v a l v u l a r t i s s u e ; i n c e l l cytoplasm a s well a s i n t h e i n t e r s t i t i u m . An accumulation o f t h e s e products was seen a t 27 Fig. 3 . Electron micrograph o f f i b r o b l a s t . P a r t of nucleus (bottom) and c y t o - plasm f i l l e d with rough endoplasmic r e t i c u l u m autophagocytic vacuoles (arrow- h e a d ) , primary lysosomes (arrow) and c e l l d e b r i s ( o r i g . mag. x 16 0 0 0 ) . Fig. 4. Electron micrograph of p a r t i a l l y degenerated f i b r o b l a s t c o n t a i n i n g primary and secondary lysosomes ( l a r g e arrow-head), s l i g h t bulging of plasma membrane i n a s s o c i a t i o n with a lysosome (small a r r o w ) , and e x p e l l e d lysosomal m a t e r i a l (small arrow-head). In t h e i n t e r f a c e abundant c o l l a g e n f i b e r s , showing c r o s s s t r i a t i o n ( l a r g e a r r o w ) , ( o r i g . mag. x 5 0 0 0 ) . t h e s u r f a c e , b u t t h e t h i c k n e s s of t h i s accumulation never exceeded one f i f t h o f t h e o r i g i n a l v a l v u l a r t h i c k n e s s . 28 F i g . 5 . E l e c t r o n micrograph of c o l l a g e n bundles, p a r t l y degenerated ( a r r o w ) , p a r t l y showing e v i d e n t c r o s s s t r i a t i o n . In t h e i n t e r f a c e secondary lysosomes of d i f f e r e n t s i z e s and shapes ( o r i g . mag. x 20 0 0 0 ) . Fig. 6 . S u r f a c e a r e a c o n t a i n i n g degenerated p l a t e l e t s ( a r r o w ) , amorphous m a t e r i a l a n d c e l l d e b r i s . Outer s u r f a c e i s covered by e l e c t r o n opaque m a t e r i a l . DISCUSSION From t h i s and e a r l i e r s t u d i e s (2, 4 , 6 , 7, 9, 1 0 , 1 4 , 15) i t i s e v i d e n t t h a t the events t a k i n g p l a c e a t the s u r f a c e o f and c e n t r a l l y i n b i o - p r o s t h e t i c v a l v u l a r tissue a r e very complex and t h a t t h e c a u s e s of t h i c k e n i n g , s h r i n k a g e a n d d y s f u n c t i o n i n t h e s e p r o s t h e s e s a r e not f u l l y understood. In t h i s s t u d y i t has been shown t h a t t r a n s i t o r y adhesion of complex p r o t e i n s a n d p l a t e l e t s 29 t a k e s p l a c e on t h e s u r f a c e of t h e t i s s u e v a l v e , b u i l d i n g u p a s u r f a c e membrane. Within t h i s membrane and i n t h e c e n t e r of t h e c u s p , breakdown products from v i a b l e f a s c i a l c e l l s could be v i s u a l i z e d both w i t h i n t h e c e l l cytoplasm a s well a s w i t h i n t h e i n t e r s t i t i a l t i s s u e . I t was thought t h a t these two p r o c e s s e s i n combination c o n t r i b u t e d t o t h e t h i c k e n i n g of t h e valve a s adhesion and d e g r a d a t i o n products occupied so much space a s v i s u a l i z e d i n t h e TEM i n v e s t i g a t i o n . T h e formation of a complex p r o t e i n l a y e r a t the s u r f a c e was thought t o prevent f u r t h e r p l a t e l e t adhesion and t h a t a t i s s u e antithrombogenic inducing f a c t o r e x i s t s i n t h e proteoglycan f r a c t i o n ( 2 ) . However, continuous adhesions of f i b r i n fragments o r thrombous m a t e r i a l seem t o t a k e p l a c e on t h e cusp s u r f a c e . These o r g a n i z e t o form a c o n n e c t i v e t i s s u e pseudointima ( 3 , 10, 1 1 , 1 2 , 1 3 ) . In o r d e r t o prevent t h i s p r o c e s s , t h e use of a n t i c o a g u l a n t s i n t h e i n i t i a l phase a f t e r valve implantation e . g . 6 weeks t o 1 0 months, has been advocated ( 1 , 5 , 9 ) . When using non-fixed b i o - p r o s t h e t i c v a l v u l a r t i s s u e i t was e v i d e n t from t h i s s t u d y t h a t breakdown products from v i a b l e c e l l s w i t h i n t h e f a s c i a l t i s s u e i t s e l f , c o n t r i b u t e t o t h e t h i c k e n i n g o f t h e cusp. This f a c t o r can only be reduced by f i x a t i o n of the v a l v u l a r t i s s u e b e f o r e i n s e r t i o n i n t o t h e h o s t by e . g . formaldehyd o r g l u t a r a l d e h y d . The l a t t e r i s now in common p r a c t i c e . and 1. 2. 3. 4. 5. 6. 7. 30 ACKNOWLEDGEMENT This work was supported by t h e Swedish National A s s o c i a t i o n a g a i n s t Heart Chest Diseases. REFERENCES Bernhard, A . , Thiede, A . , Muller-Hermelink, H - K . , Krug, A , , F i s c h e r , K. & Yankah, A . C . : Mitral v a l v e replacement w i t h autologous f a s c i a l a t a . Result of morphologic examinations. J . Thorac. Cardiovasc. Surg. 64:94, 1973. C a r p e n t i e r , A , , Relland, J . , C a r p e n t i e r , S . , Lessana, A. & Gory, G . : T i s s u e antithrombogenic inducing f a c t o r : Experimental evidence and p r a c t i c a l a p p l i c a t i o n s i n t h e c o n s t r u c t i o n of c a r c i a c v a l v e s and the a r t i f i c i a l h e a r t . Thromb. Diates. Haemorrag. 34:557, 1975. Dubiel, W.T., Johansson, L . & Willen, R . : P o s t o p e r a t i v e changes i n autologous f a s c i a l a t a h e a r t valve g r a f t s . A n n . Thorac. Surg. 15:140, 1973. Dubiel, W.T. & Willen, R . : V i a b i l i t y P r o p e r t i e s i n Autologous Fascia Lata Heart Valve G r a f t s , l a t e o b s e r v a t i o n s i n : S h i l e y European C a r c i o v a s c u l a r Conference, Chamonix, France, 1979, p . 161. , F . : Uberlagungen z u r f u n k t i o n e l l e n S t r u k t u r d e r F a s c i e . Zbl Vet- E? 9 Reie A ) 13:85, 1966. Jonescu, M.I., P a k r a s h i , B . C . , Mary, D.A.S., Bartek, I.T. & Wooler, G . H . : Long term e v a l u a t i o n of t i s s u e v a l v e s . 3 . Thorac. Cardiovasc. Surg. 68: 361, 1974. Senning, 8 . : F a s c i a l a t a replacement of a o r t i c v a l v e s . J . Thorac. Cardio- vasc. Surg. 54:465, 1967. Surg. 68:379, 1974. 9. S h i l e y European C a r d i o v a s c u l a r C o n f e r e n c e , Chamonix, F r a n c e , 1979, p. 108. 1 0 . S i l v e r , M . D . , Hudson, R . E . B . & T r i m b l e , A.S.: Morphologic o b s e r v a t i o n s on h e a r t v a l v e p r o s t h e s e s made o f f a s c i a l a t a . J . Thorac. C a r d i o v a s c . Surg. 70:360, 1975. 1 1 . S i l v e r , M . D . & T r i m b l e , A.S.: S t r u c t u r e o f a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e p r o s t h e s e s . Arch. P a t h . 93:109, 1972. 1 2 . T r i m b l e , A.S., Gunstensen, J . , S i l v e r , M . D . , A l d r i d g e , H . E . , S c h w a r t z , L . & Morch, J.E.: A o r t i c v a l v e r e p l a c e m e n t w i t h f a s c i a l a t a : an e n c o u r a g i n g l a t e s t u d y . J . Thorac. C a r d i o v a s c . Surg. 68:219, 1974. 13. Welch, W . , P o v l i e g e , P. & Promo, G . : Autologous f a s c i a l a t a c a r d i a c v a l v e r e p l a c e m e n t . Thorax 26:271 , 1971. 14. WillGn, R . , D u b i e l , W.T. & J o h a n s s o n , L . : V i a b i l i t y and s u r f a c e t w o p e r t i e s o f a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e g r a f t s . Ann.-Thorac. S u r g . ' 18':597, 1974. 15. WillGn, R . m D u b i e l , W.T. & J o h a n s s o n , L . : V i a b i l i t y p r o p e r t i e s i n a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e g r a f t s . A n n , Thorac. Surg. 19:494, 1975. Address f o r r e p r i n t s : Roger WillGn, M . D . Department o f P a t h o l o g y U n i v e r s i t y H o s p i t a l 5-221 8 5 L U N D , Sweden 31