HAL Id: jpa-00223773
https://hal.archives-ouvertes.fr/jpa-00223773
Submitted on 1 Jan 1984
HAL
is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire
HAL, estdestinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
QUANTITATIVE ELECTRONPROBE
MICROANALYSIS OF THE EPIPHYSEAL GROWTH PLATE
E.-R. Krefting, G. Lissner (willner), H. Höhling
To cite this version:
E.-R. Krefting, G. Lissner (willner), H. Höhling. QUANTITATIVE ELECTRONPROBE MICRO-
ANALYSIS OF THE EPIPHYSEAL GROWTH PLATE. Journal de Physique Colloques, 1984, 45
(C2), pp.C2-465-C2-468. �10.1051/jphyscol:19842106�. �jpa-00223773�
QUANTITATIVE ELECTRONPROBE MICROANALYSIS OF THE EPIPHYSEAL GROWTH PLATE E.-R. Krefting, G. Lissner (Willner) and H.J. Hohling
Inst. Med. Phys., Huffevstr. 68, D-4400 Munster, F.R.G.
Résumé - La concentration des différents éléments du cartilage épiphysaire a été mesurée à l'aide du microanalyseur à sonde électronique. Pour l'analyse quantitative, la formule de Hall a été utilisée. Les concentrations intra- et extracellulaires ne changent pas beaucoup dans les régions non minéralisées.
Par contre la concentration de K augmente depuis la zone de repos jusqu'à la zone hypertrophique. Les concentrations extra- cellulaires de K et de Ca sont relativement élevées par rapport aux concentrations intracellulaires. La minéralisation commence à partir des régions extracellulaires. Les concentrations m o - yennes des éléments du cartilage épiphysaire sont en bon accord avec les résultats donnés par les analyses chimiques.
Abstract - Using electronprobe microanalysis the concentration of several elements was measured in the epiphyseal growth plate.
The Hall equation was used for quantification, the standards were thin layers of freeze dried salt s o l u t i o n s . The intra- as well as the extracellular concentrations change relatively
little in the unmineralized regions. Potassium, however, demon- strated an increase going from the resting to the hypertrophic zone. Average element concentrations agree well with chemical analyses. The mineralization starts extracellularly in the vicinity of the cells in small compartments. Ca and P increase together in the course of mineralization.
The epiphyseal growth plates were taken from the tibia of 2 rats(4week old) and the ulna of 2 pigs (6 and 10 week o l d ) . Immediately after the death the growth plates were excised, partitioned into small slices and rapidly frozen in propane cooled by liquid nitrogen. Sections were cut in a cryostat (temperature of the chamber: -30 C, that of the knife and the specimen holder: -45 C ) . Each section contained all cell zones (longitudional s e c t i o n ) . The sections were placed on thin films, stretched over aluminium tubes with an open diameter of 8 mm. Another thin film was stretched over the section and slightly pressed. The specimens freeze dried in high vacuum and were finally coated with carbon. The sections were analyzed in a microprobe (CAMECA M S 4 6 ) with 3 wavelengthdispersive and an energydispersive system. The energy of the incoming electrons was 35 keV, the probe current 2.5-5 nA, the counting time 100 or 200 sec. When analysing the intra- ang extracellu- lar regions separately the probe scanned an area of lxl yum or m o r e . To analyze profiles of the elements, the probe scanned a 100 yum wide line parallel to the mineralization border, and then this line was moved slowly from the epiphyseal to the metaphyseal side of the growth plate. Sometimes a TEM (Philips 3 0 1 ) with scanning device and energy- dispersive system was used.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842106
C2-466 JOURNAL DE PHYSIQUE
F i g , 1 ( l e f t )
-
E p i p h y s e a l growth p l a t e ( t l b i a o f r a t ) . Ca= Calcium d i s t r i b u t i o n map, A E = m i c r o g r a p h t a k e n w i t h t h e a b s o r b e d e l e c t r o n s . E p i p h y s e a l s i d e a t t h e t o p . C e l l z o n e s o f t h e growth p l a t e : M = m i n e r a l i z e d , R = r e s t i n g , P= pro1 i f e r a t i n g , C = c o l u m n a r , L C = l o w e r c o l u m n a r , H= h y p e r t r o p h i c , BM= b e g i n n i n g m i n e r a l i z a t i o n . B a r = 0 . 1 mmF i g . 2 ( b e l o w )
-
a ) i n t r a - , and b ) e x t r a - c e l l u l a r e l e m e n t c o n c e n t r a t i o n s i n d i f f e r - e n t c e l l z o n e s ( F i g . 1 ) : R = r e s t i n g , P=p r o l i f e r a t i n g , C = c o l u m n a r , lC= l o w e r c o l - umnar, H = h y p e r t r o p h i c c e l l z o n e ( f r o m l e f t t o r i g h t ) . Number of m e a s u r e m e n t s : b e - tween 3 ( z o n e P) and 1 5 ( z o n e C ) . C o e f f i - c i e n t of v a r i a t i o n : between 30% ( c o n c e n t r a - t i o n h i g h e r t h a n 1 % ) and 50% ( c o n c e n t r a t i o n l e s s t h a n 0 . 5 % ) . Mass l o s s n o t t a k e n i n t o a c c o u n t .
r 1L
N a Mg P S C 1 K C a
i n ex i n ex i n e x i n e x i n e x i n ex i n ex p i g 2.0 3.4 0 . 2 0 . 1 2.2 0 . 2 1 . 0 4.5 1 . 8 2.3 1 . 0 2.2 0 . 3 0 . 6 r a t 1 . 5 3.9 0 . 2 0 . 1 2.4 0 . 3 0 . 6 3.2 1 . 3 1 . 8 3.2 2.9 0 . 2 0 . 5 Tab. 1
-
Element c o n c e n t r a t i o n s i n w e i g h t % p e r d r y mass. Mean o f t h e u n m i n e r a l i z e d z o n e s R - H. i n = i n t r a c e l l u l a r , e x = e x t r a c e l l u l a r c o n c e n - t r a t i o n s . C o e f f i c i e n t o f v a r i a t i o n 20- 30%.F o r q u a n t i f i c a t i o n t h e H a l l e q u a t i o n was u s e d . The s t a n d a r d s w e r e mix- t u r e s o f i n o r g a n i c s a l t s , s p r a y e d o n t o t h i n f i l m s and f r e e z e d r i e d ( 1 ) The e l e m e n t c o n c e n t r a t i o n s a r e g i v e n i n w e i g h t % p e r d r y mass ( g / 1 0 0 g ) . The c o e f f i c i e n t o f v a r i a t i o n i s a b o u t 20%; f o r c o n c e n t r a t i o n s l e s s t h a n 0 . 5 % i t i s a b o u t 30%. Under t h e p r o b e a b o u t 3 0 % o f t h e o r g a n i c mass i s l o s t . T h i s l o s s i s t a k e n i n t o a c c o u n t i n Tab.1 and Tab. 2.
A c c o r d i n g t o t h e m o r p h o l o g i c a l a p p e a r a n c e t h e growth p l a t e i s d i v i d e d i n t o c e l l z o n e s ( F i g . 1 ) . E x a c t b o r d e r s between them do n o t e x i s t .
from t h e e p i p h y s e a l t o t h e m e t a p h y s e a l
. . r e g i o n . Legende o f z o n e s s e e F i g . 1
.
Ca . .
STEM- m i c r o g r a p h s ( l e f t ) , Ca- d i s t r i b u t i o n maps ( r i g h t ) and x - r a y s p e c t r a o f t h e s i t e s i n d i c a t e d ( b o t t o m ) . c = c e l l s
N a Mg P C 1 K C a
z o n e R C R C R C R C R C R C
ESMA 2.0 3.4 0 . 0 5 0 . 1 0 0 . 2 0 0 . 6 7 1 . 3 0 1.60 0 . 9 3 2.20 0 . 3 4 0.35 WU 1.9 3 . 1 0.08 0.20 0 . 1 7 0 . 6 7 0 . 7 3 1 . 2 4 0 . 6 7 1 . 2 4 0 . 2 4 1 . 0 7 Q U
- -
0 . 0 6 0 . 1 5 0 . 2 0 1 . 2 1- -
0 . 8 8 1 . 6 4 0 . 2 6 2.04 Tab. 2-
Mean v a l u e s o f e l e m e n t s i n w e i g h t % p e r d r y mass i n t h e c e l l z o n e s R and C . Comparison of t h e own (EPMA= e l e c t r o n p r o b e m i c r o a n a l y - s i s ) w i t h c h e m i c a l a n a l y s e s (MU= W u t h i e r ( 2 ) ; Q U = Q u i n t ( 3 ) ). The EPMA v a l u e s t a k e i n t o a c c o u n t 3 0 % l o s s o f t h e o r g a n i c mass.F i g . 2 a ) shows t h e i n t r a - , F i g . 2 b ) t h e e x t r a c e l l u l a r c o n c e n t r a t i o n s o f t h e c e l l z o n e s R - H . I n t r a c e l l u l a r l y P d e c r e a s e s from zone R t o zone H. I n z o n e H t h e c o n t e n t o f S d e c r e a s e s , w h i l e t h a t o f K ( e s p e c i a l l y e x t r a c e l l u l a r l y ) t a s w e l l a s t h o s e o f P and Ca i n c r e a s e . P o s s i b l y t h e f i r s t m i n e r a l d e p o s i t s w e r e i n c l u d e d i n zone H . A l l t o g e t h e r t h e d i f - f e r e n c e s between t h e c e l l z o n e s a r e r e l a t i v e l y s m a l l . So Tab. 1 shows t h e means o v e r a l l c e l l z o n e s . The d i f f e r e n c e between t h e p i g and t h e r a t a r e s m a l l e x c e p t from t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f K . P i s l o c a t e d m a i n l y i n t r a c e l l u l a r e l y , I t s e x t r a c e l l u l a r c o n t e n t i s v e r y low.
The Mg c o n t e n t i s low; i t i s more c o n c e n t r a t e d i n t r a c e l l u l a r l y . O n t h e o t h e r hand t h e c o n t e n t s o f Na, S , and Ca a r e e x t r a c e l l u l a r l y h i g h e r t h a n i n t r a c e l l u l a r l y . E x c e p t from K t h i s d i s t r i b u t i o n i s a s e x p e c t e d .
F i g . 3 shows p r o f i l e s of t h e e l e m e n t s from t h e e p i p h y s e a l t o t h e m e t a p h y s e a l s i d e o f t h e growth p l a t e . Ca and P i n c r e a s e t o g e t h e r on b o t h s i d e s . K d e m o n s t r a t e s a c o n t i n u o u s i n c r e a s e from zone R t o H. The
C2-468 JOURNAL DE PHYSIQUE
c o n t e n t o f P i s r e l a t i v e l y h i g h i n zones P , C , and H , w h i l e t h a t o f Na and S i s r e l a t i v e l y s m a l l i n zone H. P r e f l e c t s t h e i n t r a - , and Na and S t h e e x t r a c e l l u l a r s p a c e i n t h e a a l y z e d a r e a (Tab. 1 ) . Because t h e
a n a l y z e d a r e a i s a b o u t 100 x 1 5 t h e amount o f t h e i n t r a - and e x t r a - c e l l u l a r s p a c e may f l u c t u a t e a t random (compare F i g . 1 ) .
Tab. 2 compares t h e m i c r o p r o b e d a t a w i t h c h e m i c a l a n a l y s e s . The mass l o s s u n d e r t h e p r o b e was t a k e n i n t o a c c o u n t . The v a l u e s a g r e e v e r y w e l l . In zone C , however, t h e c h e m i c a l l y d e t e r m i n e d c o n t e n t o f Ca ( a n d P I i s h i g h e r t h a n t h a t o f t h e m i c r o p r o b e a n a l y s i s . P r o b a b l y t h e c h e m i c a l an- a l y s i s i n c l u d e d some s m a l l m i n e r a l i z e d c o m p a r t m e n t s b e c a u s e o f t h e i r w o r s e s p a t i a l r e s o l u t i o n .
The m i n e r a l i z a t i o n s t a r t s m a i n l y i n t h e l o n g i t u d i o n a l s e p t a ( F i g . 1 ) . F i g . 4 shows t h e s i t e s o f t h e b e g i n n i n g m i n e r a l i z a t i o n a t a h i g h e r m a g n i f i c a t i o n . The m i n e r a l i z a t i o n b e g i n s i n t h e e x t r a c e l l u l a r s p a c e i n t h e v i c i n i t y o f t h e c e l l s ( F i g 4 t o p ) . The h i g h e r m a g n i f i c a t i o n o f Ca d i s t r i b u t i o n map ( m i d d l e r i g h t ) d e m o n s t r a t e s , t h a t t h e m i n e r a l i z a t i o n b e g i n s i n s m a l l c o m p a r t m e n t s . The x - r a y s p e c t r a ( b o t t o m ) show, t h a t Ca and P a r e e n l a r g e d r e a l l y i n t h e s e s m a l l compartments. The i n c r e a s e o f t h e Ca and P c o n c e n t r a t i o n a t t h e m i n e r a l i z a t i o n b o r d e r ( F i g . 3 and 4 ) i s i n a g r e e m e n t w i t h t h e a s s u m p t i o n , t h a t a p a t i t e i s d e p o s i t e d . O u r f i n d i n g s on t h e m i n e r a l i z a t i o n of a o r t a s , i n d u c e d by h i g h b l o o d p r e s - s u r e , w e r e s i m i l a r ( 4 ) .
The K c o n t e n t n e a r t h e b o r d e r o f m i n e r a l i z a t i o n i s h i g h , a l s o e x t r a - c e l l u l a r l y ( F i g . 2 ) , i n a g r e e m e n t w i t h q u a l i t a t i v e a n a l y s e s o f Boyde and S h a p i r o ( 5 ) . Some K m i g h t have d i f f u s e d d u r i n g t h e p r e p a r a t i o n o r i t m i g h t be r e q u i r e d b e s i d e s Na and Ca t o c o m p e n s a t e n e g a t i v e c h a r g e s ( t h e h i g h e x t r a c e l l u l a r S c o n t e n t m i g h t r e f l e c t a h i g h c o n t e n t o f s u l - p h a t e d m a c r o m o l e c u l e s a s c h o n d r o i t i n s u l p h a t e ) . More K m i g h t be bound i n zone L C t h a n i n zones H and BM ( 3 ) . where i t might be r e p l a c e d by Ca f o r t h e o n s e t o f m i n e r a l i z a t i o n .
The m i n e r a l i z a t i o n b e g i n s o n l y when t h e l o c a l i o n p r o d u c t o f Ca and p h o s p h a t e i s h i g h enough. M i c r o p r o b e a n a l y s i s c a n n o t d i s c r i m i n a t e be- tween i o n s and bound a t o m s . T h e r e f o r o n l y t h e p r o d u c t Ca x P o f t h e c o n c e n t r a t i o n s c a n be c a l c u l a t e d . I t i s mich h i g h e r t h a n t h e i o n p r o - d u c t n e c e s s a r y f o r t h e o n s e t o f m i n e r a l i z a t i o n ( 6 ) . Thus a l a r g e p o r - t i o n o f Ca a n d / o r p h o s p h a t e must be bound ( i . e . n o t p r e s e n t a s f r e e l y d i f f u s a b l e i o n s ) .
REFERENCES
1 KREFTING E . - R . . LISSNER G . . HUHLING H.J.. S c a n n i n a E l e c t r o n M i c r o s c .
/ i 9 8 1 / 1 1 ( i 9 8 i j 369
-
2 m R . E . , C a l c . T i s s u e Res. 4 ( 1 9 6 9 ) 20
3 OUINT P . . ALTHOFF J . , HUHLICiG H . J . , i n : O s t e o q e n e s e und Knochen-
~ ~ C ~ S ~ U ~ - ( H A C K E N B R O C H M . H . , R E F I O R - H . J . , J X G E ~ M . , e d s . ) Thieme. S t u t t g a r t ( 1 9 8 2 ) 7
4 KREFTING E . - R . , RUHRIG T . , B R U C K E R W . , A N Y A N M U E . , SCHLAKt W., DITTRICH H . , HUHLING H . J . , S c a n n i n g E l e c t r o n M i c r o s c . /1982/IV
( 1 9 8 2 ) 1617
5 B O Y D E - A . , SHAPIRO I.M., Histochem. 69 ( 1 9 8 0 ) 8 5
6 KREFTING E . - R . , BARCKHAUS R . H . , H U H n N G H.J., LISSNER G . , E l e c t r o n M i c r o s c o p y ( 1 0 t h I n t . Congr. E l e c t r . M i c r o s c . ) Vol. 3 ( 1 9 8 2 ) 387
Acknowledgement
We t h a n k t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t f o r f i n a n c i a l s u p p o r t .
