T a b l e 3 1
P r i n c i p a l modes o f p r e p a r a t i o n and s t u d y o f s a m p l e s . 1. B o i l i n g
2 . Enzym atic c l e a n i n g u s i n g Terg-azym e, 7 - 5 gms i n 1 l i t r e , 5 -1 0 d a y s , change o f s o l u t i o n e v e r y d a y , 5 0°C.
3. T o p o g r a p h i c a l d o c u m e n ta tio n (P h o to g ra p h y )
4 . 4mm s q u a r e b e ^ from M cIII c o n t a i n i n g e i t h e r m e d ia l o r l a t e r a l n o t c h , and AP s l i c e s w ith w a t e r c o o le d slow s p e e d diamond saw, c l e a n e d f o r SEM.
Figure 3*1 Top - D i f f e r e n t t y p e s o f t h e c o n d y l a r f r a c t u r e o f t h e t h i r d m e t a c a r p a l bone: a ) I n c o m p l e t e , b) C o m p l e t e - n o n d i s p l a c e d , c) C o m p l e t e - d i s p l a c e d ( a f t e r R ick e t a l 1983 and R ig g s 1 9 9 0 ) . B o t t o m - A c e n t r a l 4 mm t h i c k m e d i o - l a t e r a l s e c t i o n o f a t h i r d m e t a c a r p a l bone from a c o n d y l a r f r a c t u r e c a s e (6 y e a r ) .
The p h o t o g r a p h i s t a k e n w i t h a 35nim c a m e ra w i t h a 80mm m acro l e n s . W hite s q u a r e s = 1 cm.
Figure 3•1
5 . 4mm c r o s s - s e c t i o n s and i n t e r m e d i a t e s l i c e s o f t h e beeims, c l e a n e d . 6. M ounting i n DPX. 7 . P o l a r i s e d l i g h t m ic ro s c o p y 8 . A u t o f l u o r e s c e n c e mode CSLM 9 . S u b c h o n d ra l ( s u b - a r t i c u l a r c a r t i l a g e ) bone b l o c k s , c l e a n e d . 10. Hydrogen p e r o x i d e , 4 ti m e s d i l u t e d , 2 h r / 2% 24 h r s . 11. Gold s p u t t e r c o a t i n g 12. * C o n v e n t i o n a l ' SE SEM 1 3. S t a i n w i t h B r i l l i a n t S u l p h a f l a v i n e i n 90% e t h a n o l . 14. PMMA embedding and p o l i s h i n g .
1 5. Carbon c o a t i n g , SEM BSE.
1 6. CSLM o f PMMA embedded beams and b l o c k s .
The m a t e r i a l s s t u d i e d c o n s i s t e d o f T horo u g h b red r a c e h o r s e lim b s w i t h i n h o u r s o f e u t h a n a s i a o f a n i m a l s d e s t r o y e d b e c a u s e o f s p o n ta n e o u s f r a c t u r e s d u r i n g r a c i n g o r t r a i n i n g . Thus M c III from I6 a n i m a l s , two w i t h M c I I I a n d two w i t h a h i n d l i m b P I f r a c t u r e , w e re c o l l e c t e d and s t o r e d w i t h s k i n i n t a c t a t -2 0 * . Bones w ere d i s s e c t e d as soon a s p o s s i b l e and sawn w h i l e deep f r o z e n .
Each M cIII was sawn m e d i o - l a t e r a l l y i n t o 3 o r 4 p i e c e s u s i n g a b a n d saw . The t h i c k n e s s o f t h e m i d d l e p i e c e was 4mm. E x p e r i m e n t s h av e b een c o n d u c te d t o d i s c o v e r a s a t i s f a c t o r y c l e a n i n g r e g im e which w i l l remove a l l s o f t t i s s u e e l e m e n t s , i n c l u d i n g p e r i o s t e a l and marrow t i s s u e , bone c e l l s and f a t . I n i t i a l l y some s am p les were b o i l e d i n a d e t e r g e n t s o l u t i o n (Nunamaker e t a l 1989) f o r a p p r o x i m a t e l y 15 h o u r s . The c l e a n e d b o n e , d e v o i d o f s o f t t i s s u e s , s t i l l c o n t a i n e d b l o o d and f a t d e e p w i t h i n t h e t i s s u e . O t h e r s a m p l e s w e r e t r e a t e d b y a c o m m e r c i a l enzym e c l e a n i n g d e t e r g e n t (B o y d e 1 9 8 4 ; R i g g s 1 9 9 0 ) d e s i g n e d f o r c l e a n i n g s u r g i c a l i n s t r u m e n t s ( T e r g - a - z y m e , A l d r i c h c h e m ic a l C o ., M ilwaukee, USA). However, u n l i k e p r e v i o u s p r o c e d u r e s , t h e bone s am p les h ad t o be t r e a t e d f o r a l o n g e r p e r i o d a t 5 0 *C
f o r 1 0 - 1 5 d a y s w i t h a s o l u t i o n (7*5 gm i n 1 l i t r e ) c h a n g e e v e r y a l t e r n a t e d a y . T h e l a t e r t r e a t m e n t , c o m b i n e d w i t h m e c h a n i c a l d e b r id e m e n t u s i n g a w a t e r j e t , and d e f a t t i n g i n a c e t o n e h a s p ro v e d t o be s a t i s f a c t o r y i n g e n e r a t i n g sam p les w i t h r e l a t i v e l y u n changed bone m a t r i x w ith c l e a n s u r f a c e s a v a i l a b l e , f o r exam ple, f o r SEM s t u d y .
Figure 3«2
P l a n o f s e c t i o n i n g o f t h e bone a t t h e s i t e o f t h e f r a c t u r e .
a ) The 4 mm s q u a r e beams ( t o p ) c o n t a i n s t h e n o t c h , t h e s i t e o f t h e i n i t i a t i o n o f t h e f r a c t u r e .
b) The l o w e r p i c t u r e shows t h e b l o c k s , o b t a i n e d by c u t t i n g t h e beam a t e v e r y 4 mm i n t h e w a t e r c o o l e d diam ond saw . The im a g e s a r e t a k e n w i t h a 35nim cam era w ith a 80mm macro l e n s .
Figure 3 . 2
The a r c h i t e c t u r e o f t h e t r a b e c u l a r b o n e i n M c I I I s a m p l e s was s t u d i e d u s i n g p l a n e p a r a l l e l 4mm s l i c e s ( f i g u r e s 3 . 2 a , 3 * 3 a b c ) and s q u a r e s e c t i o n l o n g i t u d i n a l beams (Boyde e t a l 1989. 1990b) c u t u s i n g a s lo w s p e e d , w a t e r c o o l e d diam ond saw ( B u e h l e r i s o m e t ) t o m i n i m i s e damage t o more d e l i c a t e t r a b e c u l a r e l e m e n t s . The l o n g i t u d i n a l beams w e re p r e p a r e d s u c h t h a t e a c h c o n t a i n e d e i t h e r t h e m e d i a l o r t h e l a t e r a l n o tc h o f t h e a r t i c u l a r s u r f a c e ( f i g . 3*2 a ) . T h ese s p e c im e n s w e re d o c u m e n te d by p h o t o g r a p h i n g a l l s u r f a c e s a t a l l l e v e l s (35mm c a m e r a w i t h 80mm m a cro l e n s ) . S a m p l e s w e r e e x a m in e d b y SEM a f t e r g o l d s p u t t e r c o a t i n g .
Some w ere s t a i n e d (9 a n i m a l s : 6yG, 7 y , 8 y , l l y , 1 2 y - 2 , l 8 y - 2 , u n k . ) w ith t h e s t a b l e f l u o r e s c e n t dye b r i l l i a n t s u l p h a f l a v i n e i n 90% e t h a n o l , d e h y d r a t e d i n a c e t o n e , a n d e m b e d d e d i n p o l y - m e t h y l - m e t h a c r y l a t e : PMMA (Boyde an d J o n e s 1 9 8 3 ; Boyde 1 9 8 4 ; Boyde e t a l 1 9 8 6; R eid and Boyde 1987; Boyde e t a l 1 9 9 2 ). PMMA monomer h a s a low v i s c o s i t y and p e n e t r a t e s w e l l i n t o b o n e. I n i t i a l l y vacuum em bedding was t r i e d , b u t d i s c o n t i n u e d b e c a u s e b u b b l e s c o u l d n o t b e a v o i d e d . However, s u c c e s s f u l embedments was a c h i e v e d by s e r i a l i n f i l t r a t i o n i n PMMA w hen 95% ( b y v o l u m e ) d i s t i l l e d m e t h y l m e t h a c r y l a t e (BDH C h e m ic a l s , UK) was c o - p o l y m e r i s e d w ith 5% (by volume) s t y r e n e (BDH) a t 37°C , and d e s t a b i l i s e d w i t h 0.2% by w e i g h t 2 , 2 - a z o - b i s (2 m e t h y l p r o p i o n i t r i l e ) . S t y r e n e was u s e d t o r e n d e r t h e e m b e d d in g r e s i n more s t a b l e t o e l e c t r o n beam bom bardm ent (Boyde 1984; Boyde e t a l I9 8 6; Boyde e t a l 1992) as PMMA i s u n s t a b l e u n d e r e l e c t r o n bombardm ent. To overcome t h e problem e n c o u n t e r e d i n em bedding t h e l o n g s l e n d e r beam s, b o i l i n g tu b e s were u s e d a s d i s p o s a b l e em bedding ’ c a p s u l e s ’ . P o l i s h e d s u r f a c e s a r e r e q u i r e d f o r c o n f o c a l LM a n d BSE SEM i m a g i n g m o d e s . P ro b le m s w ere e n c o u n t e r e d i n p o l i s h i n g a l l f o u r s i d e s o f e a c h beam, b u t i t seems s a t i s f a c t o r y t o p o l i s h two o r t h r e e m u t u a l l y o r t h o g o n a l