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A morphological study of the formation of the dentino-enamel junction in the rat molar : the role of.. 1989

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A MORPHOLOGICAL STUDY OF THE FORMATION OF THE DENTINO-ENAMEL JUNCTION IN THE RAT MOLAR: THE ROLE OF DENTIN IN ENAMEL FORMATION. B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES (Department of Anatomy) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1989 £/) Bruce W i l l i a m Robinson, 1989 By BRUCE WILLIAM ROBINSON i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT This t h e s i s i s a morphological study d e t a i l i n g the i n i t i a l formation of the dentino-enamel j u n c t i o n (DEJ) i n the r a t molar. I t was undertaken to determine how enamel a p a t i t e c r y s t a l s are i n i t i a t e d during tooth development. The DEJ was i n v e s t i g a t e d along i t s developmental gradient so that both the s p a t i a l and temporal sequences of dentin and enamel m i n e r a l i z a t i o n could be observed. For t h i s i n v e s t i g a t i o n both conventional and s e l e c t e d - a r e a d a r k - f i e l d e l e c t r o n microscopy were a p p l i e d . The r e s u l t s of t h i s study i n d i c a t e t h a t enamel c r y s t a l s a r i s e by e p i t a x i a l growth from preformed dentin c r y s t a l s . This i s supported by the temporal r e l a t i o n s h i p beween dentin and enamel where dentin m i n e r a l i z a t i o n preceeds enamel m i n e r a l i z a t i o n and the intimate s p a t i a l r e l a t i o n s h i p between the a p a t i t e c r y s t a l s of dentin and enamel at both the newly formed and the more mature dentino-enamel j u n c t i o n . These r e s u l t s a l s o suggest t h a t the t e r m i n a l portions of collagen f i b r i l s i n the pre-dentin matrix undergo some a l t e r a t i o n p r i o r to m i n e r a l i z a t i o n which may be r e l a t e d t o t h e i r f u n c t i o n i n presenting dentin a p a t i t e to the enamel matrix. The i n t e r r e l a t o n s h i p between both the i n o r g a n i c and organic matrices of dentin and enamel are presented i n a working model and discussed i n l i g h t of the mechanisms by which m i n e r a l i z e d dentin may promote the formation of enamel c r y s t a l s . TABLE OF CONTENTS Page Number Abstract i i A bbreviations iv/ L i s t of Figures v Acknowledgements v i H i s t o r i c a l Review 1 I n t r o d u c t i o n 25 M a t e r i a l s and Methods 28 Results 35 Discussion 64 Conclusions 70 References 72 i v ABBREVIATIONS BF b r i g h t f i e l d BL basal lamina BM basement membrane Ca2+ calcium DAJ dentino-ameloblast j u n c t i o n DEJ dentino-enamel j u n c t i o n ECM e x t r a c e l l u l a r matrix EDTA ethylenediaminetetra-acetic a c i d EM e l e c t r o n microscopy GAG glycosaminoglycan hrs hours LC lead c i t r a t e LM l i g h t microscopy min minutes mis m i l l i l i t r e s nm nanometers PTA '• phosphotungstic a c i d P phosphate RER rough endoplasmic re t i c u l u m SADF selected-area dark f i e l d TEM transmission e l e c t r o n microscopy UA uranyl acetate urn micrometers V LIST OF FIGURES Figure T i t l e Page Number 1. Overview of the formation of the 37 dentino-enamel j u n c t i o n . 2. I n i t i a t i o n and subsequent m i n e r a l i z a t i o n 42 of dentin. 3. Changes i n collagen as the dentino- 45 enamel j u n c t i o n forms. 4. Sequential steps of enamel m i n e r a l i z a t i o n . 48 5. The enamel sheath. 50 6. C r y s t a l c o n t i n u i t y - newly formed 53 ' dentino-enamel j u n c t i o n . 7. C r y s t a l o r g a n i z a t i o n i n dentin. 55 8. C r y s t a l c o n t i n u i t y - more mature enamel, 58 9. Schematic representation of dentino-enamel 61 j u n c t i o n formation. vi ACKNOWLEDGEMENTS I would l i k e to thank my family f o r supporting me through t h i s degree and f o r t h e i r l o n g s u f f e r i n g when i t appeared that I was i n gradual s t u d i e s as opposed to graduate s t u d i e s . I would e s p e c i a l l y l i k e to thank Robyn McCreery f o r g i v i n g me joy and the w i l l to continue studying, and to Dr. Arsenault f o r h i s patience and m o t i v a t i o n a l cracks of the whip when mine f a l t e r e d . A debt of g r a t i t u d e i s al s o owed to my f e l l o w graduate students who have allowed me to share not only ideas with them, but a l s o my f r i e n d s h i p . To Dr. Slonecker, Dr. Ovalle and the other f a c u l t y who have taught me i n courses and con t r i b u t e d to improving my research s k i l l s , I thank you as w e l l . This research has been supported by a grant to Dr. A.L. Arsenault from the Medical Research Council of Canada. 1 HISTORICAL REVIEW This t h e s i s deals with the events of m i n e r a l i z a t i o n i n tooth dentin and enamel i n r e l a t i o n to the formation of the dentino-enamel j u n c t i o n (DEJ). At t h i s j u n c t i o n there e x i s t s an i n t i m a t e s p a t i a l r e l a t i o n s h i p between these two e x t r a c e l l u l a r matrices (ECMs). This r e l a t i o n s h i p i s important because i t provides a f i r m attachment f o r enamel to dentin enabling the tooth to maintain i t s s t r u c t u r a l i n t e g r i t y while involved i n m a s t i c a t i o n . New evidence of t h i s r e l a t i o n s h i p at the DEJ i s explored at the e l e c t r o n microscopic l e v e l i n t h i s t h e s i s , but i n order to f u l l y understand how t h i s r e l a t i o n s h i p i s formed and what components are i n v o l v e d , a comprehensive review i s r e q u i r e d . The review presented here provides background information about the c e l l u l a r i n t e r a c t i o n s l e a d i n g up to the formation of the DEJ, the biochemistry of the organic components of dentin and enamel, how m i n e r a l i z a t i o n i s thought to occur, and s p e c i f i c information about a p a t i t e c r y s t a l . DENTIN 1 ) C e l l u l a r Development The c e l l s responsible f o r e l a b o r a t i o n of the dentin matrix are odontoblasts. Their d i f f e r e n t i a t i o n from mesenchymal c e l l s i n the stomodeum i s thought to r e s u l t from genetic predetermination and i n t e r a c t i o n with the o v e r l y i n g e p i t h e l i u m during odontogenesis ( T h e s l e f f and Hurmerinta,1981; S l a v k i n et a l . , 1984). The mesenchymal c e l l s i n the m a x i l l a r y and mandibular arches 2 t h a t give r i s e to odontoblasts are c o l l e c t i v e l y r e f e r r e d to as ectomesenchyme. Unlike the surrounding mesenchymal c e l l s , the ectomesenchyme i s thought to have a r i s e n from the neural c r e s t . Along the arches these c e l l s form d i s t i n c t c l u s t e r s beneath the o r a l e p i t h e l i u m (Weston 1981). Studies of carbohydrate histochemisty have shown that the presence of N-acetyl-D-glucosamine i n the basement membrane (BM) causes the condensation of the ectomesenchymal c e l l s (Lau and Ruch, 1983). The BM a l s o has been i m p l i c a t e d as having a r o l e i n the d i f f e r e n t i a t i o n of the ectomesenchymal c e l l s i n t o pre-odontoblasts (Ruch, 1984). A recent study by B l o t t n e r and Linder (1987) using peroxidase conjugated to d i f f e r e n t l e c t i n s , demonstrated progressive stages of odontogenesis based on the s p a t i a l and temporal expression of three sugar residues a s s o c i a t e d with the BM. With i n v i t r o s t u d i e s K o l l a r and B a i r d (1970) grew molar ectoderm on i n c i s o r mesenchyme and v i c e versa observing i n both cases th a t the mesenchyme c o n t r o l l e d both the morpho- and c y t o d i f f e r e n t i a t i o n of the ectoderm to form the mesemchymal type of tooth germ. This i n d u c t i v e c o n t r o l by mesenchyme i s not l i m i t e d to o r a l ectoderm, as the mesenchyme can form tooth germs when associated with ectoderm from other s i t e s on the body. While the mesenchyme seems to orchestrate the formation of tooth germs, i t cannot d i f f e r e n t i a t e without an a s s o c i a t e d ectoderm ( T h e s l e f f and Hurmerinta, 1981 ). • E l e c t r o n microscopic s t u d i e s have demonstrated a prominent BM between the e p i t h e l i a l and the mesenchymal c e l l s (Bernard, 1972; S l a v k i n and Bringas, 1976). Biochemical s t u d i e s have c h a r a c t e r i z e d t h i s dental BM to be composed of type IV c o l l a g e n , heparin sulphate, 3 l a m i n i n , s u l p h a t e d p r o t e o g l y c a n s and f i b r o n e c t i n s i m i l a r t o t h a t o f o t h e r BM ( K e f a l i d e s , 1 9 7 3 ; M a r t i n e z - H e r n a n d e z e t a l . , 1 9 8 1 ; B i r e m b a u t e t a l . , 1 9 8 3 ) . D u r i n g t h e t i m e when mesenchymal c e l l s o r g a n i z e a l o n g t h e BM, i t s f i b r o n e c t i n c o n t e n t i n c r e a s e s ( T h e s l e f f e t a l . , 1981 ) . T h i s f i n d i n g s u p p o r t s t h e o r i e s f o r t h e r o l e o f f i b r o n e c t i n i n a t t a c h i n g mesenchymal c e l l s t o t h e BM whereby they unde rgo a p o l a r i z a t i o n o f c e l l u l a r o r g a n e l l e s and b e g i n t o i n c r e a s e t h e amount o f s e c r e t o r y a c t i v i t y . Mesenchymal c e l l s t h a t r e a c h t h i s s t a g e o f d i f f e r e n t i a t i o n i n t he t o o t h germ a r e r e f e r r e d t o a s p r e - o d o n t o b l a s t s . H i s t o l o g i c a l l y , t h e s e c e l l s a r e o r g a n i z e d i n t o a l a y e r o f p o s t m i t o t i c , low c o l u m n a r c e l l s w i t h b a s a l l y l o c a t e d n u c l e i . Each c e l l has an i n c r e a s e d amount o f rough e n d o p l a s m i c r e t i c u l u m (RER) w h i c h a l i g n s p a r a l l e l t o t he l o n g a x i s o f t h e c e l l , a c e n t r a l l y l o c a t e d G o l g i a p p a r a t u s and numerous f r e e r i b o s o m e s and m i t o c h o n d r i a ( R e i t h 1 9 6 8 ) . Mesenchyme c y t o d i f f e r e n t i a t i o n i s a l s o a s s o c i a t e d w i t h an i n c r e a s e d s y n t h e s i s and r e l e a s e o f s u l p h a t e d g l y c o s a m i n o g l y c a n s (GAGS) and g l y c o p r o t e i n s i n t o t h e ECM a p i c a l t o p r e - o d o n t o b l a s t s ( T h e s l e f f and H u r m e r i n t a 1 9 8 1 ) . T r a n s - f i l t e r s t u d i e s a r e used t o d e m o n s t r a t e t he r e q u i r e m e n t o f a c l o s e p r o x i m i t y between p r e - o d o n t o b l a s t c e l l s and t h e BM o f t h e enamel e p i t h e l i u m f o r o d o n t o b l a s t d i f f e r e n t i a t i o n t o o c c u r . In t h e s e s t u d i e s p r e - o d o n t o b l a s t and enamel e p i t h e l i a l c e l l s a r e e n z y m a t i c a l l y s e p a r a t e d and p l a c e d on o p p o s i t e s i d e s o f f i l t e r p a p e r , and a l l o w e d t o c o n t i n u e t o d i f f e r e n t i a t e ( T h e s l e f f and H u r m e r i n t a , 1981 ) . O n l y t h e mesenchymal c e l l s whose a p i c a l c e l l p r o c e s s e s a r e c l o s e l y a s s o c i a t e d w i t h t h e 8M o f t h e e p i t h e l i u m d i f f e r e n t i a t e i n t o p r e - o d o n t o b l a s t s . E x p e r i m e n t s where t he BM o f d e v e l o p i n g t o o t h germs were d i s r u p t e d w i t h 4 v i t a m i n A o r t u n i c a m y c i n , t h e mesenchyme d i d no t d i f f e r e n t i a t e ( H u r m e r i n t a e t a l . , 1980 ; T h e s l e f f and P r a t t , 1 9 8 0 ) . The p r e - o d o n t o b l a s t , a f t e r i n i t i a l d i f f e r e n t i a t i o n , b e g i n s t o s e c r e t e a p r e d e n t i n m a t r i x w h i c h i s composed o f c o l l a g e n t y p e s I, I I I , II/, U, p r o t e o g l y c a n s , g l y c o p r o t e i n s , serum p r o t e i n s and m a t r i x v e s i c l e s ( L i n d e , 1 9 8 5 ) . D u r i n g t h i s t i m e t he p r e - o d o n t o b l a s t c e l l c o n t i n u e s t o d i f f e r e n t i a t e becoming t a l l e r and b e i n g pushed f u r t h e r away f rom t h e BM. The o n l y p o r t i o n o f t h e c e l l t h a t r e m a i n s i n c l o s e p r o x i m i t y t o t h e BM i s i t s a p i c a l c e l l p r o c e s s . 2) P r e d e n t i n / D e n t i n O r g a n i c M a t r i x P r e d e n t i n and d e n t i n d i f f e r w i t h r e s p e c t t o t he a b s e n c e o r p r e s e n c e o f a m i n e r a l component . O t h e r d i f f e r e n c e s between t h e o r g a n i c m a t r i c e s o f p r e d e n t i n and d e n t i n a r e o b s e r v e d b i o c h e m i c a l l y a s changes i n t he t y p e s and c o n c e n t r a t i o n s o f n o n - c o l l a g e n o u s p r o t e i n s ( B u t l e r 1 9 8 4 a ) . Ano the r d i f f e r e n c e o b s e r v e d a t t he e l e c t r o n m i c r o s c o p i c (EM) l e v e l i s t h e p r e s e n c e o r a b s e n c e o f m a t r i x v e s i c l e s ; t h e s e v e s i c l e s a r e o n l y p r e s e n t i n t h e p r e d e n t i n p r i o r t o i t s i n i t i a l m i n e r a l i z a t i o n ( B e r n a r d , 1972 ; K a t c h b u r i a n , 1 9 7 3 ) . Once t he i n i t i a l l a y e r o f d e n t i n i s f o r m e d , m a t r i x v e s i c l e s a r e no t o b s e r v e d i n e i t h e r t h e p r e d e n t i n o r t h e d e n t i n . A) C o l l a g e n A number o f t y p e s o f c o l l a g e n have been i d e n t i f i e d i n t h e p r e - d e n t i n m a t r i x , t hey i n c l u d e : t y p e I, I I I , I V , and V , bu t o n l y c o l l a g e n t y p e s I and V a r e f o u n d i n d e n t i n ( B u t l e r 1 9 8 4 b ) . Type I i s t h e most abundan t p r o t e i n , mak ing up 8 0 - 9 0 % o f bo th m a t r i c e s ( L i n d e , 1 9 8 5 ) . I t a p p e a r s randomly a r r a n g e d a s i n d i v i d u a l f i b r i l s t h r o u g h o u t t h e m a t r i c e s o r as o r g a n i z e d b u n d l e s , r e f e r r e d t o as von K o r f f f i b r e s , 5 e x t e n d i n g f r o m between t he p r e - o d o n t o b l a s t c e l l p r o c e s s e s t o t h e BM i n p r e - d e n t i n . Bo th g roups o f f i b r i l s show t h e c h a r a c t e r i s t i c 64 nm r e p e a t e d b a n d i n g p a t t e r n by EM a n a l y s i s ( R e i t h , 1 9 6 8 ; W i t t a k e r e t a l . , 1 9 7 2 ; L a r s s o n and B l o o m , 1 9 7 3 ; W i g g l e s w o r t h e t a l . , 1 9 8 6 ) . The f o r m a t i o n o f c o l l a g e n t y p e I f r o m o d o n t o b l a s t c e l l s has been s t u d i e d u s i n g t r i t i a t e d p r o l i n e a u t o r a d i o g r a p h y ( w e i n s t o c k and L e b l o n d , 1 9 7 4 ) . Each c o l l a g e n m o l e c u l e i s made up o f a t r i p l e h e l i x ; t h e s e m o l e c u l e s a r e o r g a n i z e d i n a s t a g g e r e d f a s h i o n and a r e h e l d t o g e t h e r by c r o s s l i n k s . A t the ends o f t h e m o l e c u l e t h e r e i s a s p a c e w h i c h , b e c a u s e o f t he r e g u l a r s t a g g e r o f m o l e c u l e s , l i n e s up l a t e r a l l y i n t h e f i b r i l c r e a t i n g t he r e g u l a r c r o s s - b a n d i n g p a t t e r n seen a t t h e EM l e v e l when s t a i n e d w i t h heavy m e t a l s (Hodge and P e t r u s k a , 1 9 6 3 ) . The l a t e r a l s p a c e s o f t he c o l l a g e n f i b r i l a r e r e f e r r e d t o a s t h e gap z o n e s and t h e a r e a between them i s r e f e r r e d t o as t he o v e r l a p z o n e . T h e r e has been much d e b a t e o v e r t h e e x i s t e n c e o f von K o r f f f i b r e s i n t h e p r e - d e n t i n m a t r i x ; Ten C a t e e t a l . (1970) d e n i e d t h e i r e x i s t e n c e b e c a u s e t he f i b r e s t h a t a p p e a r a t t he l i g h t m i c r o s c o p i c (LM) l e v e l when i m p r e g n a t e d w i t h s i l v e r c o r r e s p o n d t o g round s u b s t a n c e and n o t ' c o l l a g e n when v iewed a t the EM l e v e l . T h i s c o n t r o v e r s y may l i e i n t h e d e f i n i t i o n o f a von K o r f f f i b r e . R e i t h ( 1 9 6 8 ) , U i t t a k e r e t a l . (1972) and W i g g l e s w o r t h e t a l . (1986) d e f i n e t h e s e f i b r e s based a t t h e EM l e v e l a s o r g a n i z e d b u n d l e s o f c o l l a g e n f i b r i l s , whereas Ten C a t e (1970) i s d e f i n i n g them as a r g e n t o p h i l i c f i b r e s o b s e r v e d a t t h e l i g h t m i c r o s c o p i c (LM) l e v e l . These f i b r e s may have a r o l e i n o r i e n t i n g t h e enamel m i n e r a l i z a t i o n because t h e y r u n p e r p e n d i c u l a r t o t he BM i n t h e p r e - d e n t i n m a t r i x ( W i g g l e s w o r t h e t a l . , 1 9 8 6 ) . The von K o r f f f i b r e s can be 1 0 0 - 2 5 0 nm i n d i a m e t e r and s t r e t c h 6 t h e w i d t h o f p r e d e n t i n whereas i n d i v i d u a l c o l l a g e n f i b r i l s w i t h i n d e n t i n a r e 2 5 - 5 0 nm ( J o h a n s e n , 1 9 6 4 ) . D u r i n g m i n e r a l i z a t i o n d e n t i n c r y s t a l s seem t o r a d i a t e a l o n g b o t h t h e s e o r g a n i z a t i o n s o f c o l l a g e n t y p e I f i b r i l s - t h e i r p r o p o s e d f u n c t i o n w i l l be d i s c u s s e d l a t e r . Q u a n t i t a t i v e i n f o r m a t i o n on changes i n c o l l a g e n f i b r i l morpho logy has no t been p r e s e n t e d a t t h i s t i m e . C o l l a g e n t y p e \1 has been i d e n t i f i e d i n p r e d e n t i n and d e n t i n . I t i s l o c a t e d i n t h e s e two m a t r i c e s n e a r o r a s s o c i a t e d w i t h c e l l u l a r c o m p o n e n t s ; howeve r , i t s f u n c t i o n i s n o t known ( B u t l e r , 1 9 8 4 b ) . The p r e s e n c e o f c o l l a g e n t y p e I I I i n t h e p r e d e n t i n m a t r i x i s somewhat c o n t r o v e r s i a l . G a r a n t and Cho (1985) have s u g g e s t e d t h a t t h i s c o l l a g e n makes up t he r e t i c u l a r ne twork o f a p e r i o d i c f i b r i l s p r e s e n t b e n e a t h t h e BM. C o l l a g e n t y p e I I I may have a r o l e i n t he i n h i b i t i o n o f m i n e r a l i z a t i o n as i t i s f o u n d i n l a r g e amounts i n c o n n e c t i v e t i s s u e t h a t does no t m i n e r a l i z e , and i n t h e p o o r l y m i n e r a l i z e d d e n t i n o f p a t i e n t s w i t h o s t e o g e n e s i s i m p e r f e c t a ( B u t l e r , 1984b ; Sauk e t a l . , 1 9 8 0 ) . Norma l m i n e r a l i z a t i o n o n l y b e g i n s a f t e r the d i s r u p t i o n and r e m o v a l o f t h e BM and i t s a s s o c i a t e d c o m p o n e n t s . An i m m u n o l a b e l l i n g s t u d y , h o w e v e r , has shown t h a t c o l l a g e n t y p e I I I i s no t p r e s e n t i n e i t h e r p r e - d e n t i n o r d e n t i n ( C o u r n i l e t a l . , 1 9 7 9 ) . Those who s u p p o r t t h e p r e s e n c e o f c o l l a g e n t y p e I I I i n p r e d e n t i n d i s m i s s t h i s s t u d y a s no t b e i n g s e n s i t i v e enough , t h u s l e a v i n g t h e c o n t r o v e r s y u n r e s o l v e d ( G a r a n t and C h o , 1 9 8 5 ) . C o l l a g e n t y p e 11/ has been d e m o n s t r a t e d a s p a r t o f t h e BM be tween t h e p r e - a m e l o b l a s t c e l l s and t h e p r e - d e n t i n m a t r i x ( T h e s l e f f e t a l . , 1 9 8 1 ) . In t he BM t h i s c o l l a g e n f o rms a s t r u c t u r a l backbone upon w h i c h t he o t h e r components a t t a c h ( T i m p l e t a l . , 1 9 8 1 ) . C o l l a g e n 7 t y p e IV p r e s e n t i n i s o l a t e d f r a c t i o n s o f p r e - d e n t i n d i s a p p e a r s a f t e r t h e BM has been d i s r u p t e d and removed ( B u t l e r , 1 9 8 4 b ) . B) I M o n - c o l l a q e n o u s p r o t e i n s The n o n - c o l l a g e n o u s p r o t e i n s a r e a h e t e r o g e n e o u s g roup o f m o l e c u l e s t h a t make up t h e amorphous b a c k g r o u n d m a t e r i a l between c o l l a g e n f i b r i l s . T h e i r i d e n t i t i e s and f u n c t i o n s have been d e t e r m i n e d by h i s t o c h e m i c a l and b i o c h e m i c a l s t u d i e s . They appea r t o be s i m i l a r t o t h o s e f o u n d i n t he o r g a n i c m a t r i x o f bone ( L i n d e , 1 9 8 4 ) . The ma jo r g roups o f n o n - c o l l a g e n o u s p r o t e i n s i n c l u d e : p r o t e o g l y c a n s , y - c a r b o x y g l u t a m a t e - c o n t a i n i n g p r o t e i n s , p h o s p h o p r o t e i n s , a c i d i c g l y c o p r o t e i n s and serum p r o t e i n s ( B u t l e r , 1 9 8 4 a ; L i n d e , 1 9 8 4 ) . D u r i n g t h e m i n e r a l i z a t i o n o f d e n t i n , t h e s e g r o u p s have d i f f e r e n t r o l e s a n d , a s e x p e c t e d , a r e p r e s e n t i n v a r i o u s c o n c e n t r a t i o n s and l o c a t i o n s . The p r o t e o g l y c a n s o f d e n t i n a r e m o l e c u l e s c o n t a i n i n g a p r o t e i n c o r e w i t h one o r two GAG c h a i n s a t t a c h e d t o i t , t h e y can be f o u n d i n t h e i n t e r f i b r i l l a r r e g i o n s o f b o t h p r e d e n t i n and d e n t i n ( T a k a g i e t a l . , 1 9 8 1 ) . E x t r a c t i o n and b i o c h e m i c a l a n a l y s i s o f t h e s e m o l e c u l e s show t h a t c h o n d r o i t i n - 4 - s u l p h a t e was t h e m a j o r GAG a s s o c i a t e d w i t h t h e p r o t e i n c o r e . O the r GAGs p r e s e n t i n l e s s amounts i n c l u d e c h o n d r o i t i n - 6 - s u l p h a t e , dermatan s u l p h a t e , h e p a r i n s u l p h a t e and h y a l u r o n i c a c i d ( J o n e s and L e a v e r , 1 9 7 4 ; R a h e m t u l l a e t a l . , 1 9 8 4 ) . A r e c e n t e x p e r i m e n t u s i n g a h i g h i r o n d i a m i n e EM t e c h n i q u e , w h i c h s p e c i f i c a l l y demons ra tes s u l p h a t e d p r o t e o g l y c a n s , and e n z y m a t i c d i g e s t i o n c o n f i r m s e a r l i e r r e p o r t s o f ' c h o n d r o i t i n s u l p h a t e i n d e n t i n a s w e l l a s b e i n g a s s o c i a t e d w i t h t h e BM p r i o r t o d e n t i n f o r m a t i o n . H e p a r i n s u l p h a t e was a l s o a s s o c i a t e d w i t h t h e BM and may have some 8 r o l e i n t h e c y t o d i f f e r e n t i a t i o n o f o d o n t o b l a s t s (Kugaya an F u r u h a s i , 1987) . E l e c t r o n m i c r o s c o p i c a u t o r a d i o g r a p h y u i i t h l a b e l l e d s u l p h a t e i o n s d e m o n s t r a t e d two m e t a b o l i c a l l y d i f f e r e n t p o o l s o f p r o t e o g l y c a n s , t h e f i r s t i s i n t he p r e - d e n t i n m a t r i x and t h e o t h e r i s s e c r e t e d j u s t i n a d v a n c e o f m i n e r a l i z a t i o n i n d e n t i n ( S u n d s t r o m 1 9 7 1 ) . At t h e t i m e o f m i n e r a l i z a t i o n t h e amount o f s u l p h a t e d g l y c o p r o t e i n s d e c r e a s e s , w h i c h s u g g e s t s t h a t t h e s e p r o t e o g l y c a n s and GAGs have a r o l e i n t h e r e g u l a t i o n o f m i n e r a l i z a t i o n ( L i n d e , 1 9 8 5 ) . A n o t h e r r o l e may be i n r e g u l a t i n g c o l l a g e n f i b r i l f o r m a t i o n and i n b i n d i n g f i b r i l s t o g e t h e r ( Q b r i n k , 1 9 7 3 ) . The y - c a r b o x y g l u t a m i c a c i d c o n t a i n i n g p r o t e i n s a r e s m a l l - 47 t o 53 amino a c i d r e s i d u e s . One o f t h e s e p r o t e i n s was f i r s t i s o l a t e d by L i n d e e t a l . (1982) i n d e n t i n , b u t had p r e v i o u s l y been p u r i f i e d f rom bone and named o s t e o c a l c i n ( P r i c e e t a l . , 1 9 7 6 ) . R e c e n t i m m u n o l a b e l i n g e x p e r i m e n t s show t h a t t h i s c a l c i u m b i n d i n g p r o t e i n does n o t p l a y a r o l e i n i n i t i a l m i n e r a l i z a t i o n b e c a u s e m a t r i x v e s i c l e s a r e n o t l a b e l l e d w i t h a n t i - o s t e o c a l c i n . L a b e l l e d RER and o d o n t o b l a s t p r o c e s s e s s u g g e s t t h a t i t i s s y n t h e s i z e d by o d o n t o b l a s t s and a c c u m u l a t e s i n d e n t i n a f t e r m i n e r a l i z a t o n o c c u r s (de V r i e s e t a l . , 1988) . I t s f u n c t i o n i s unknown. A n o t h e r g roup o f n o n - c o l l a g e n o u s p r o t e i n s a r e t h e p h o s p h o p r o t e i n s . The ma jo r amino a c i d s o f t h e p h o s p h o p r o t e i n s a r e s e r i n e and a s p a r t a t e wh i ch a r e h i g h l y p h o s p h o r y l a t e d ( D i m u z i o and V e i s , 1 9 7 8 ; R i c h a r d s o n e t a l . , 1 9 7 8 ) . These p h o s p h o p r o t e i n s , a l s o r e f e r r e d t o a s p h o s p h o r y n s , a r e o n l y f o u n d i n t he d e n t i n m a t r i x , h a v i n g been s e c r e t e d a t t h e m i n e r a l i z a t i o n f r o n t ( M a c D o u g a l l e t a l . , 9 1 9 8 3 ) . T h e i r i m p o r t a n c e i n t he p r o c e s s o f m i n e r a l i z a t i o n i s s u g g e s t e d by t h e i r a b s e n c e i n d e n t i n o g e n e s i s i m p e r f e c t a I I where t h e d e n t i n i s p o o r l y m i n e r a l i z e d ( T a k a g i e t a l . , 1 9 8 3 ) . I s o l a t e d p h o s p h o r y n s have been shown by r o t a r y shadow e l e c t r o n m i c r o s c o p y t o b i n d Ca2+ i n t h e p r e s e n c e o f c a l c i u m c h l o r i d e ( C o c k i n g - J o h n s o n e t a l . , 1 9 8 3 ) . B i n d i n g domains on p h o s p h o r y n s , p r o p o s e d i n a model by L e e e t a l . (1977) s u g g e s t t h a t t h e p h o s p h o r y n s a r e i n v o l v e d i n n u c l e a t i n g a p a t i t e and p e r h a p s c o n t r o l t h e s i z e o f d e n t i n a p a t i t e c r y s t a l s . A r e c e n t s t u d y by L u s s i e t a l . (1988) d e m o n s t r a t e d t h a t p u r i f i e d p h o s p h o r y n s f r e e i n a c a l c i u m and p h o s p h a t e (CaP) s o l u t i o n i n h i b i t e d t h e p r e c i p i t a t i o n o f a p a t i t e , bu t when t h e p h o s p h o r y n s were c p v a l e n t l y a t t a c h e d t o a g a r o s e beads i n t he same s o l u t i o n a p a t i t e f o r m a t i o n was i n d u c e d . O n l y low amounts o f p h o s p h o r y n s were r e q u i r e d t o i n d u c e c r y s t a l f o r m a t i o n i f a t t a c h e d t o a g a r o s e b e a d s . P h o s p h o r y n s may have b o t h an i n h i b i t o r y and an i n d u c t i v e r o l e i n d e n t i n m i n e r a l i z a t i o n . The p r e s e n c e o f o t h e r a c i d i c g l y c o p r o t e i n s has been d e t e r m i n e d , bu t o t h e r t h a n t h e i r m o l e c u l a r w e i g h t s , few s t u d i e s have p u r i f i e d and c h a r a c t e r i z e d them ( B u t l e r , 1 9 8 4 a ; L i n d e , 1984 , 1 9 8 5 ) . Serum p r o t e i n s have a l s o been r e p o r t e d , t h e s e i n c l u d e a l b u m i n , t r a n s f e r r i n , I g G , I gM, and IgA (Thomas and L e a v e r , 1 9 7 5 ) . T h e i r f u n c t i o n i n d e n t i n i s unknown a t t h e p r e s e n t t i m e . C) M a t r i x V e s i c l e s EM s t u d i e s have d e m o n s t r a t e d m a t r i x v e s i c l e s t o be p r e s e n t i n t h e p r e - d e n t i n m a t r i x . Such v e s i c l e s a r e f ound benea th t h e b a s a l l a m i n a i n t h e amorphous m a t e r i a l between c o l l a g e n f i b r i l s . K a t c h b u r i a n (1977) d e s c r i b e d them a s b e i n g 3 0 - 3 0 0 nm i n d i a m e t e r w i t h a l i m i t i n g t r i l a m i n a r p l a s m a membrane 7 -8 nm w i d e . . They v a r y i n shape 10 and i n t h e e l e c t r o n d e n s i t y o f t h e i r c o n s t i t u e n t s and a r e t h o u g h t t o be f o rmed by b u d d i n g o f f f rom o d o n t o b l a s t p r o c e s s e s ( S l a v k i n e t a l . , 1 9 7 2 ; S i s k a and P r o v e n z a , 1 9 7 2 ) . S e r i a l s e c t i o n s and f r e e z e f r a c t u r e s o f t h e p r e - d e n t i n m a t r i x hav/e d e m o n s t r a t e d t h a t t he m a t r i x v e s i c l e s a r e d i s t i n c t f r om o d o n t o b l a s t p r o c e s s e s even though they a p p e a r t o have s i m i l a r c o n t e n t s ( K a t c h b u r i a n , 1 9 7 3 , K a t c h b u r i a n and S e v e r s , 1 9 8 2 ) . M a t r i x v e s i c l e s a r e p r e s e n t i n t he p r e - d e n t i n m a t r i x f r o m t h e p o i n t where t h e mesenchyme become p r e - o d o n t o b l a s t s u n t i l t h e m a t r i x i s m i n e r a l i z e d ( B e r n a r d , 1 9 7 2 ; L a r s s o n and B l o o m , 1 9 7 3 ) . W i t h i n t h i s a r e a S l a v k i n e t a l . (1972) r e c o g n i z e d d i s t i n c t t y p e s o f m a t r i x v e s i c l e s b a s e d upon t h e i r s i z e and e l e c t r o n d e n s i t y . K a t c h b u r i a n and S e v e r (1982) a l s o r e c o g n i z e d a h e t e r o g e n e i t y between m a t r i x v e s i c l e s when t h e y compared t he number o f i n t r a m e m b r a n e p r o t e i n p a r t i c l e s o f e a c h v e s i c l e r e v e a l e d by f r e e z e f r a c t u r e r e p l i c a t i o n . A f u n c t i o n p r o p o s e d t o be a s s o c i a t e d w i t h one group o f m a t r i x v e s i c l e s i s t h e i n i t i a t i o n o f d e n t i n m i n e r a l i z a t i o n . T h i s was s u g g e s t e d i n i t i a l l y because e l e c t r o n - d e n s e m a t e r i a l s i m i l a r i n shape t o ' the a p a t i t e c r y s t a l s o b s e r v e d i n d e n t i n was f o u n d i n some v e s i c l e s ( S l a v k i n e t a l . , 1 9 7 2 ; K a t c h b u r i a n , 1 9 7 3 ; B e r n a r d , 1 9 7 3 ; E i senman and G l i c k , 1 9 7 2 ) . O t h e r e v i d e n c e f o r t h i s f u n c t i o n shows t h a t i s o l a t e d m a t r i x v e s i c l e s f rom p r e - d e n t i n use ATP t o c o n c e n t r a t e c a l c i u m ( G r a n s t o n , 1 9 8 4 ) . As t h e r e a r e v e s i c l e s w h i c h do no t c o n t a i n m a t r i x v e s i c l e s n o t a l l m a t r i x v e s i c l e s a r e t h o u g h t t o be i n v o l v e d i n t h e i n i t i a t i o n o f m i n e r a l i z a t i o n i n p r e - d e n t i n ( S l a v k i n e t . a l . , 1 9 7 2 ) . A n o t h e r f u n c t i o n o f m a t r i x v e s i c l e s i s i n the t r a n s p o r t o f e n z y m e s , a c t i n g a s e x t r a c e l l u l a r l y s o s o m e s ( S l a v k i n e t a l . , 1 9 7 2 ; 11 K a t c h b u r i a n , 1 9 7 3 ) . E v i d e n c e d e m o n s t r a t i n g t h e p r e s e n c e o f a c i d p h o s p h a t a s e w i t h i n t h e s e v e s i c l e s s u p p o r t s t h i s f u n c t i o n ( L a r s s o n , 1 9 7 3 ) . M a t r i x v e s i c l e s may a l s o have a r o l e i n t h e t r a n s p o r t a t i o n o f s t r u c t u r a l m o l e c u l e s i n t o t h e m a t r i x f r om t h e o d o n t o b l a s t s , and t h e y may p r o v i d e a means o f c o m m u n i c a t i o n be tween t h e p r e - o d o n t o b l a s t and t h e p r e - s e c r e t a r y a m e l o b l a s t p r i o r t o c a l c i f i c a t i o n ( S l a v k i n e t a l . , 1 9 7 2 ) . These l a s t two f u n c t i o n s a r e more d i f f i c u l t t o s u b s t a n t i a t e . The p r e s e n c e o f m a t r i x v e s i c l e s i s no t l i m i t e d t o p r e d e n t i n , t h e y a r e a l s o f o u n d i n c a r t i l a g e , b o n e , and c a l c i f y i n g e x t r a - s k e l e t a l t i s s u e s where t h e y a p p e a r p r i m a r i l y t o be i n v o l v e d i n t h e i n i t i a t i o n o f m i n e r a l i z i n g a c o l l a g e n o u s e x t r a c e l l u l a r m a t r i x ( B o n u c c i , 1 9 8 4 ) . A r s e n a u l t and H u n z i k e r (1988) were t h e f i r s t t o s p e c i f i c a l l y d e t e r m i n e t h e p r e s e n c e o f a p a t i t e w i t h i n m a t r i x v e s i c l e s . T h i s was a c h e i v e d by u s i n g s e l e c t e d - a r e a da rk f i e l d (SADF) i m a g i n g on e p i p h y s e a l c a r t i l a g e p r e s e r v e d by h i g h p r e s s u r e f r e e z i n g . 3) M i n e r a l i z a t i o n EM s t u d i e s have d e s c r i b e d t h e p r o c e s s o f m i n e r a l i z a t i o n o f p r e - d e n t i n a s b e g i n n i n g i n membrane-bounded m a t r i x v e s i c l e s c o n t a i n i n g e l e c t r o n - d e n s e c r y s t a l - l i k e s t r u c t u r e s . The c r y s t a l s i n c r e a s e i n number w i t h i n t h e v e s i c l e and t h e n e x c e e d s p a s t t h e v e s i c l e membrane, g r o w i n g e p i t a x i a l l y t h r o u g h t he a f i b r i l l a r g round s u b s t a n c e . These r a d i a t i n g c r y s t a l s appea r as i s l a n d s o r l o c i o f e l e c t r o n d e n s i t y i n u n s t a i n e d s e c t i o n s ( H a y a s h i , 1 9 8 4 ) . C r y s t a l g row th c o n t i n u e s t h r o u g h t h e g round s u b s t a n c e t o c o l l a g e n f i b r i l s w h i c h becomes i m p r e g n a t e d w i t h t h e m i n e r a l . As m i n e r a l i z a t i o n p r o g r e s s e s , t h e s e p a r a t e l o c i c o a l e s c e and fo rm a c o n t i n u o u s l a y e r d e s c r i b e d a s m a n t l e d e n t i n ( B e r n a r d , 1 9 7 2 ; S i s k a and P r o v e n z a 1 9 7 2 ; H a y a s h i , 1984 ; B o c c h i e r i e t 12 a l . , 1 9 8 5 ) . S e v e r a l mechanisms have been p r o p o s e d f o r t h e r o l e o f m a t r i x v e s i c l e s i n i n i t i a t i n g m i n e r a l i z a t i o n i n d e n t i n . K a t c h b u r i a n (1973) p r o p o s e d t h a t t h e s e v e s i c l e s o r i g i n a t e f r o m o d o n t o b l a s t s e i t h e r b e i n g s y n t h e s i z e d i n t h e i r c y t o p l a s m and t h e n e x o c y t o s e d o r by b u d d i n g o f f o d o n t o b l a s t c e l l p r o c e s s e s . I t was assumed t h a t t h e v e s i c l e s c o n t a i n e d Ca and P i o n s i n s i m i l a r c o n c e n t r a t i o n s t o t h a t f o u n d i n o d o n t o b l a s t s , h o w e v e r , w h i l e i n the p r e d e n t i n t h e p l a s m a membrane o f a v e s i c l e u n d e r g o e s a change i n i t s p e r m e a b i l i t y b e c a u s e i t i s no l o n g e r a s s o c i a t e d w i t h a c e l l and t h e r e f o r e i s n o t r e n e w e d . The change i n membrane p e r m e a b i l i t y r e s u l t s i n t h e c r e a t i o n o f a s u p e r - s a t u r a t e d i o n i c c o n d i t i o n t h a t p r e c i p i t a t e s a p a t i t e c r y s t a l . A n o t h e r a u t h o r s u g g e s t s t h a t t h e m a t r i x v e s i c l e s c o n c e n t r a t e i o n s by ATP/CA2+ pumps d e t e c t e d - i n membranes o f i s o l a t e d v e s i c l e s ( G r a n s t r o m , 1 9 8 4 ) . P h o s p h a t e head g r o u p s o f membrane l i p i d s may be r e s p o n s i b l e f o r b i n d i n g Ca i o n s c o n c e n t r a t e d w i t h i n t h e v e s i c l e s and p r e c i p i t a t i n g a p a t i t e c r y s t a l s w h i c h appea r t o fo rm on t h e i r i n n e r membrane s u r f a c e (Eanes and H a i l e r , 1 9 8 5 ) . T h e r e i s an i n c r e a s e i n c r y s t a l number and s i z e w i t h i n t he v e s i c l e s u n t i l t h e v e s i c l e can no l o n g e r c o n t a i n them, i t b u r s t s and c r y s t a l g row th e x t e n d s i n t o t he s u r r o u n d i n g p r e d e n t i n . M a t r i x v e s i c l e s do no t have a r o l e i n s u b s e q u e n t m i n e r a l i z a t i o n a s t h e y a r e no t o b s e r v e d i n p r e d e n t i n a f t e r m a n t l e d e n t i n i s fo rmed (E i senman and G l i c k , 1 9 7 2 ) . C o n t i n u e d m i n e r a l i z a t i o n i s t h o u g h t "to be due t o a h i g h c o n c e n t r a t i o n o f Ca and P , b o t h f r e e and p r o t e i n - b o u n d i n t h e p r e d e n t i n m a t r i x (Munhoz and L e b l o n d , 1 9 7 4 ; B u t l e r , 1 9 8 4 ) . The c a l c i u m c o n c e n t r a t i o n o f t h e d e n t i n m a t r i x i s c o n t r o l l e d 13 by t h e o d o n t o b l a s t s . S t u d i e s u s i n g p y r o a n t i m o n a t e show t h a t c a l c i u m i s t a k e n up by o d o n t o b l a s t s f rom l a t e r a l l y and b a s a l l y l o c a t e d c a p i l l a r i e s and d e p o s i t e d a p i c a l l y i n t h e e x t r a c e l l u l a r m a t r i x ( A p p l e t o n and M o r r i s , 1 9 7 9 ) . The pathway o f c a l c i u m t h r o u g h t h e o d o n t o b l a s t c e l l s i s c o n f i r m e d by t h e p r e s e n c e o f t i g h t j u n c t i o n s be tween c e l l s wh i ch l a n t h a n u m , an i o n s i m i l a r i n s i z e t o c a l c i u m , c a n n o t t r a v e r s e ( B i s h o p , 1 9 8 5 ) . D u r i n g c o n t i n u e d m i n e r a l i z a t i o n , c a l c i u m i o n s a r e a l s o s u p p l i e d by t he s u p p o r t i n g l a y e r s o f a m e l o b l a s t s - t h e s t r a t u m i n t e r m e d i a and s t e l l a t e r e t i c u l u m . The p r e s e n c e o f phospha te i n t h e d e n t i n m a t r i x a s s o c i a t e d w i t h p h o s p h o r y n s has been d i s c u s s e d i n r e l a t i o n t o i t s p r o p o s e d r o l e i n a p a t i t e f o r m a t i o n . Wot much i s known a b o u t t h e mechanism o f p h o s p h a t e t r a n s p o r t a t i o n i n t o t h e p r e - d e n t i n m a t r i x b u t x - r a y m i c r o a n a l y s i s and m i c r o - c h e m i c a l a n a l y s i s has d e m o n s t r a t e d i t s p r e s e n c e i n r e l a t i v e l y h i g h c o n c e n t r a t i o n s ( H o h l i n g and Fromme, 1 9 8 4 ) . I n i t i a l c r y s t a l s fo rmed w i t h i n and a r o u n d m a t r i x v e s i c l e s a p p e a r t o be n e e d l e - l i k e w i t h an a v e r a g e w i d t h o f 2 . 6 nm. As m i n e r a l i z a t i o n p r o g r e s s e s , c r y s t a l s a r e d e s c r i b e d as more p l a t e - l i k e a n d ' measure abou t 5 -6 nm i n w i d t h ( H a y a s h i , 1 9 8 3 , and F r a n k , 1 9 8 0 ) . T h i s i n c r e a s e i n w i d t h i s t h o u g h t t o r e s u l t f rom t h e f u s i o n o f t h e i n i t i a l n e e d l e - l i k e c r y s t a l s ( H a y a s h i , 1 9 8 4 ) . F u s i o n has a l s o been i m p l i c a t e d i n t h e p r e f e r e n t i a l m i n e r a l i z a t i o n o f c o l l a g e n f i b r i l s . The i n i t i a l m i n e r a l i z a t i o n o f c o l l a g e n was t h o u g h t o n l y t o o c c u r I n gap zones o f c o l l a g e n f i b r i l s ( G l i m c h e r , 1 9 5 9 ) . I t has s i n c e been d e m o n s t r a t e d w i t h c o n v e n t i o n a l EM and SADF i m a g i n g t h a t m i n e r a l i z a t i o n can o c c u r i n bo th t h e gap and t h e o v e r l a p z o n e s , a l t h o u g h - t o a l e s s e r e x t e n t i n t h e l a t t e r ( A r s e n a u l t , 1988 ; 1 9 8 9 ; 14 H o h l i n g e t a l . , 1 9 7 1 ) . The r o l e o f c o l l a g e n i n m i n e r a l i z a t i o n may be i n p r o v i d i n g an e n v i r o n m e n t wh i ch t r a p s c a l c i u m and p h o s p h a t e i o n s g i v i n g an i o n pathway f o r c r y s t a l g row th t o f o l l o w . 4 ) D e n t i n C r y s t a l s E l e c t r o n d i f f r a c t i o n and x - r a y d i f f r a c t i o n p a t t e r n s show d e n t i n c r y s t a l s t o be t h e i n s o l u b l e c a l c i u m p h o s p h a t e s a l t , h y d r o x y a p a t i t e ( T r a u t z , 1966 ; P o s n e r and Tannenbaum, 1 9 8 4 ) . C h e m i c a l a n a l y s i s o f t h e c r y s t a l s show t h a t t h e y a r e no t p u r e h y d r o x y a p a t i t e , b u t c o n t a i n c a r b o n a t e , w a t e r and t r a c e i n o r g a n i c c o n s t i t u e n t s ( P e t e r s s o n e t a l . , 1 9 7 8 ) . These i m p u r i t i e s a l t e r t h e mo la r r a t i o o f c a l c i u m t o p h o s p h a t e w h i c h i s a p p r o x i m a t e l y 1 .67 i n mature d e n t i n and s l i g h t l y l e s s i n f o r m i n g d e n t i n ( P o s n e r and Tannenbaum, 1 9 8 4 ) . D e n t i n c r y s t a l s , l i k e o t h e r c r y s t a l s i n m i n e r a l i z e d t i s s u e s , have a u n i t c e l l w i t h t h e c h e m i c a l f o r m u l a C a 1 0 ( P 0 4 ) 6 ( 0 H ) 2 and d i m e n s i o n s d e l i n i a t e d by x - r a y and n e u t r o n d i f f r a c t i o n o f 0 . 9 4 2 nm i n t h e a - and b - a x i s and 0 . 6 8 8 nm i n t h e c - a x i s ( G l a s , 1 9 6 2 ) . These u n i t c e l l s a r e p a r a l l e l p i p e d i n shape and a c t a s a t e m p l a t e f o r t h e a t o m i c a r r a n g e m e n t o f t h e who le c r y s t a l . They l i n e up so t h a t t he c - a x i s i s t h e ' l o n g a x i s o f t h e c r y s t a l and t h e a - a x i s i s t h e t h i c k n e s s . B e c a u s e t h e s e u n i t c e l l s a r e a r r a n g e d i n an e q u a l , r e p e a t e d p a t t e r n t h e y c a n be u s e d t o d e r i v e b o t h t h e o v e r a l l s i z e and o r i e n t a t i o n o f c r y s t a l s i n d e n t i n . A number o f s t u d i e s have measured t h e d i m e n s i o n s o f ma tu re d e n t i n c r y s t a l s , w h i c h a r e t i g h t l y r e g u l a t e d , t o be 20 t o 35 nm l o n g and 5 - 6 nm t h i c k and w ide ( J e n s e n , and R o l l e r , 1 9 4 8 ; T r a u t z e t a l . , 1 9 5 3 ; F r a n k , 1 9 8 0 ) . The re have been few s t u d i e s t o d e l i n e a t e t h e o r g a n i z a t i o n o f c r y s t a l s w i t h i n d e n t i n . 15 ENAMEL Enamel i s u n l i k e any other mammalian mi n e r a l i z e d t i s s u e because i t i s formed by ectodermally derived c e l l s , and does not con t a i n c o l l a g e n , or matrix v e s i c l e s and i s more h i g h l y m i n e r a l i z e d . 1) C e l l u l a r Development So f a r only the embryonic epithelial/mesenchymal i n t e r a c t i o n s l e a d i n g to dentin m i n e r a l i z a t i o n have been reviewed. This i n t e r a c t i o n i s a l s o required f o r the o r a l epithelium of the stomodeal membrane to become se c r e t o r y ameloblasts and form enamel. The underlying mesenchyme i s r e s p o n s i b l e f o r inducing the i n v a g i n a t i o n of the stomodeal ectoderm and i t s eventual m o r p h o - d i f f e r e n t i a t i o n forming the enamel organ of the tooth germ. M o r p h o - d i f f e r e n t i a t i o n r e s u l t s from an increased m i t o t i c a c t i v i t y of the c e l l s of the stratum germinitivum l a y e r . Once morphological d i f f e r e n t i a t i o n has ceased, the t i s s u e o r g a n i z a t i o n resembles the tooth shape described h i s t o g i c a l l y as the b e l l stage ( R e i t h , 1967). As ' described i n the dentin review, the inner enamel epi t h e l i u m , which r e s t s upon a BM, induces the mesenchyme c e l l s on the other side to d i f f e r e n t i a t e i n t o odontoblasts and begin to secrete the collagenous pre-dentin matrix. The BM has been i m p l i c a t e d i n the c y t o - d i f f e r e n t i a t i o n of odontoblasts, but i t s a s s o c i a t i o n with the predentin matrix induces p o l a r i t y i n the inner enamel e p i t h e l i u m . This p o l a r i z a t i o n r e s u l t s i n a d i s t a l l y l o c a t e d nucleus, c e n t r a l l y l o c a t e d G o l g i apparatus, an increase of mitochondria located a p i c a l l y and d i s t a l l y , and many ci s t e r n a e of RER running p a r a l l e l to the long 16 a x i s o f t h e s e c e l l s ( K a l l e n b a c h , 1 9 7 2 ) . The p r e - d e n t i n m a t r i x c o n t a i n s c o l l a g e n a s e s t h a t may degrade t h e BM ( S o r g e n t e e t a l . , 1977) w h i c h i s t h e n removed by t h e p r e - s e c r e t o r y a m e l o b l a s t c e l l s . EM e v i d e n c e o f p i n o c y t o t i c v e s i c l e s a l o n g t h e a p i c a l b o r d e r o f a m e l o b l a s t s s u g g e s t s t h e i r r o l e i n t he e n d o c y t o s i s o f t he b r o k e n down BM ( B e r n a r d , 1 9 7 2 ; K a l l e n b a c h , 1 9 7 6 ) . F o l l o w i n g t h e d i s r u p t i o n o f t h e BM, p r o x i m a l p r o c e s s e s o f t he a m e l o b l a s t s a r e o b s e r v e d i n d i r e c t c o n t a c t w i t h o d o n t o b l a s t p r o c e s s e s ; t h i s e v e n t may i n d u c e t h e a m e l o b l a s t t o b e g i n t he r e m o v a l o f t he BM ( S l a v k i n and B r i n g a s , 1 9 7 6 ) . C o n t a c t be tween t h e s e two c e l l s and t h e r e m o v a l o f t h e BM a r e c o n c u r r e n t w i t h t h e a p p e a r a n c e o f e l e c t r o n - d e n s e s e c r e t o r y v e s i c l e s n e a r t h e a p i c a l c e l l b o r d e r w i t h i n t h e p r e - s e c r e t o r y a m e l o b l a s t . These s e c r e t o r y v e s i c l e s a r e no t r e l e a s e d u n t i l d e n t i n m i n e r a l i z a t i o n r e a c h e s t h e d e n t i n o - a m e l o b l a s t j u n c t i o n (OAJ) ( B e r n a r d , 1 9 7 2 ) . When m i n e r a l i z a t i o n r e a c h e s t h i s p o i n t , enamel m a t r i x i s r e l e a s e d by e x o c y t o s i s d e s c r i b e d as m o d i f i e d m e r o c r i n e s e c r e t i o n ( S i m m e l i n k , 1 9 8 2 ; N a n c i e t a l . , 1 9 8 7 ) . The e l e c t r o n - d e n s e enamel m a t r i x b a t h e s m i n e r a l i z e d c o l l a g e n f i b r i l s o f d e n t i n ( B e r n a r d 1 9 7 2 ) . Soon a f t e r t h e m a t r i x i s r e l e a s e d i t m i n e r a l i z e s , w i t h o u t t h e a i d o f m a t r i x v e s i c l e s . The i n i t i a l p l a t e - l i k e enamel c r y s t a l s a r e o b s e r v e d t o be fo rmed a t t h e d e n t i n o - e n a m e l j u n c t i o n and grow t owa rd t h e a m e l o b l a s t c e l l ( R e i t h 1 9 6 7 ) . I t has been s u g g e s t e d by B e r n a r d (1972) t h a t enamel c r y s t a l s a r e j u s t e x t e n s i o n s o f d e n t i n c r y s t a l s . 2) Enamel P r o t e i n s The newly fo rmed enamel m a t r i x has an o r g a n i c component and a m i n e r a l componen t . The ma jo r p a r t o f t h e o r g a n i c component i s t h e enamel p r o t e i n s w h i c h make up 20-30% o f i n i t i a l m a t r i x . These 17 p r o t e i n s a r e c h a r a c t e r i z e d by t h e i r amino a c i d c o m p o s i t i o n , m o l e c u l a r w e i g h t , and on t h e i r b i o c h e m i c a l p r o p e r t i e s , i t i s now a g r e e d t h a t t h e r e a r e two g r o u p s o f enamel p r o t e i n s - t h e a m e l o g e n i n s and e n a m e l i n s . The a m e l o g e n i n s a r e the s m a l l e r m o l e c u l a r w e i g h t p r o t e i n s r a n g i n g f rom 5 -40 k d , bu t t h e y make up 80-90% o f t h e o r g a n i c m a t r i x i n i n i t i a l enamel (Te rm ine e t a l . , 1 9 8 0 ) . I s o l a t i o n o f t h i s g roup o f p r o t e i n s w i t h BM g u a n i d i n e HC1 and a n a l y s i s o f t h e i r amino a c i d c o n t e n t shows them t o be r i c h i n p r o l i n e w i t h l e s s e r amounts o f g l u t a m i c a c i d , l e u c i n e , and h i s t i d i n e ( E a s t o e , 1 9 7 9 , S l a v k i n e t a l . , 1 9 8 2 ) . I t has been s u g g e s t e d t h a t t h e s e p r o t e i n s make u p . a l a r g e p o r t i o n o f t h e s t i p p l e d m a t e r i a l i n t o w h i c h t he enamel c r y s t a l s grow d u r i n g t he i n i t i a l m i n e r a l i z a t i o n o f e n a m e l . The a m e l o g e n i n s a r e n o t t h o u g h t , h o w e v e r , t o be c l o s e l y a s s o c i a t e d w i t h t h e c r y s t a l s b e c a u s e t h e y a r e e a s i l y e x t r a c t e d l e a v i n g the c r y s t a l s i n t a c t (Te rm ine e t a l . , 1 9 7 9 ; L y a r u u e t a l . , 1 9 8 2 ) . I m m u n o l a b e l l i n g o f t h e enamel m a t r i x has shown t h a t t h e a m e l o g e n i n s occupy t he m a t r i x be tween enamel c r y s t a l s , w h i c h s u g g e s t s t h e y have a space f i l l i n g r o l e i n t h e enamel m a t r i x ( H a y a s h i e t a l . , 1 9 8 5 ) . In f r e e z e f r a c t u r e o f enamel t h e a m e l o g e n i n s have a g l o b u l a r a p p e a r a n c e w h i c h i s d i s p e r s e d t h r o u g h o u t t h e m a t r i x between c r y s t a l s . T h e i r a p p e a r a n c e was s i m i l a r t o o t h e r n o n - s t r u c t u r a l p r o t e i n s l i k e a l b u m i n and o v a l b u m i n a f t e r i n c u b a t i o n w i t h e x t r a c t e d enamel c r y s t a l s and f r e e z e f r a c t u r e ( B a i and Warshawsky , 1 9 8 5 ) . O t h e r e v i d e n c e f o r t h e i r s p a c e f i l l i n g r o l e comes f rom s t u d i e s t h a t c o r r e l a t e changes i n t h e p r o t e i n c o n t e n t o f enamel a s i t m a t u r e s . I t a p p e a r s t h a t p r o t e a s e s a r e s e c r e t e d by t he a m e l o b l a s t s t h a t s e l e c t i v e l y b reakdown 18 a m e l o g e n i n p r o t e i n s w h i c h a r e t h e n s e l e c t i v l y removed ( O v e r a l l and L i m e b a c k , 1 9 8 8 ) . T h i s l o s s o f a m e l o g e n i n s c o r r e s p o n d s w i t h an i n c r e a s e i n m i n e r a l i z a t i o n . These changes o f t h e a m e l o g e n i n s a r e w e l l documented (Te rm ine e t a l . , 1979 , R o b i n s o n e t a l . , 1 9 7 7 , 1 9 8 3 , S a s a k i e t a l . , 1 9 8 2 , R o b i n s o n and K i r k h a m , 1 9 8 5 , B a i and Ularshawsky, 1 9 8 5 ) . S t u d i e s c o m p a r i n g a m e l o g e n i n s be tween s p e c i e s show t h i s g roup o f p r o t e i n s i s c o n s e r v e d ( S l a v k i n e t a l . , 1 9 8 2 ; R o b i n s o n and K i r k h a m , 1 9 8 5 ) . The o t h e r ma jo r g roup of p r o t e i n s i n t h e enamel m a t r i x a r e t h e e n a m e l i n s . These a r e l a r g e m o l e c u l a r w e i g h t a c i d i c g l y c o p r o t e i n s 6 0 - 7 5 kd t h a t a r e a n t i g e n i c a l l y d i s t i n c t f r om a m e l o g e n i n s (Rosenb loom e t a l . , 1 9 8 6 ) . T h i s group o f p r o t e i n s i s t h o u g h t t o be i n i t i m a t e l y a s s o c i a t e d w i t h enamel c r y s t a l s b e c a u s e t h e y can o n l y be e x t r a c t e d a f t e r d e m i n e r a l i z a t i o n o f t he enamel w i t h EDTA (Term ine e t a l . , 1 9 7 9 ) . A n a l y s i s o f t h e amino a c i d c o m p o s i t i o n o f t h e e n a m e l i n s shows them t o be r i c h i n a s p a r t i c a c i d , s e r i n e , g l u t a m i c a c i d , and g l y c i n e . C o n f i r m a t i o n o f c o n t a c t between t he e n a m e l i n s and enamel c r y s t a l s was d e m o n s t r a t e d by a n t i g e n i c l a b e l l i n g o f enamel c r y s t a l s w i t h a n t i - e n a m e l i n ( H a y a s h i e t a l . , 1 9 8 6 ) . The e n a m e l i n s a r e t h o u g h t t o f o rm an enamel s h e a t h t h a t can be o b s e r v e d e l e c t r o n m i c r o s c o p i c a l l y i n d e m i n e r a l i z e d e n a m e l . T h i s s h e a t h s t r e t c h e s f r o m the d e n t i n o e n a m e l j u n c t i o n (DEJ) t o t he a m e l o b l a s t c e l l s u r f a c e i n a l l s t a g e s o f enamel m i n e r a l i z a t i o n ( J e s s e n , 1 9 6 8 ; G u s t a u s e n and S i l n e s s , 1969 ; Y a n a g i s a w a and IMylen, 1 9 7 9 ; A r s e n a u l t and R o b i n s o n , 1 9 8 9 ) . I t i s b e l i e v e d t h a t t h e s e p r o t e i n s have a f u n c t i o n i n m i n e r a l i z a t i o n . An e a r l y x - r a y d i f f r a c t i o n s t u d y showed t h a t t h e d e m i n e r a l i z e d enamel m a t r i x gave a c r o s s b e t a d i f f r a c t i o n p a t t e r n 19 s u g g e s t i n g a s t r u c t u r a l component t o t h i s m a t r i x ( G l i m c h e r e t a l . , 1 9 6 1 ) . A more r e c e n t s t u d y by T raub e t a l . (1985) c o n f i r m e d t h a t t h e e n a m e l i n s a r e r e s p o n s i b l e f o r a b e t a - p l e a t e d s h e e t c o n f o r m a t i o n c l o s e l y a s s o c i a t e d w i t h enamel c r y s t a l s . T h i s s t u d y a l s o showed t h a t t h e enamel s h e a t h a f t e r d e m i n e r a l i z a t i o n c o u l d be r e m i n e r a l i z e d by b e i n g i n c u b a t e d i n a c a l c i u m c h l o r i d e b a t h . T h i s s u g g e s t e d t h a t t h e s h e a t h may hav/e a r o l e i n the n u c l e a t i o n o f enamel h y d r o x y a p a t i t e c r y s t a l s . F u r t h e r e v i d e n c e f o r t h i s i s p r e s e n t e d i n t h i s s t u d y by T raub e t . a l . ( 1 9 8 5 ) , a s t h e phospha te backbone o f t h e p r o p o s e d s h e a t h s t r u c t u r e c o r r e s p o n d s t o t h e u n i t c e l l d i m e n s i o n s o f h y d r o x y a p a t i t e . E l e c t r o n s p e c t r o s c o p i c image a n a l y s i s a l s o d e m o n s t r a t e s t h a t t h e e n a m e l i n s h e a t h i s i n v o l v e d i n t h e c o - l o c a l i z a t i o n o f c a l c i u m and p h o s p h a t e t h e r e b y p l a y i n g a r o l e i n t he g r o w t h o f enamel c r y s t a l s ( A r s e n a u l t and R o b i n s o n , 1 9 8 9 ) . U n l i k e t he a m e l o g e n i n group o f p r o t e i n s , t h e e n a m e l i n p r o t e i n s r e m a i n r e l a t i v e l y c o n s t a n t t h r o u g h o u t enamel m a t u r a t i o n , wh i ch a r e t h e m a j o r p r o t e i n g roup i n f u l l y formed enamel (Te rm ine e t a l . , 1 9 8 0 ) . A r e c e n t s t u d y by Menanteau e t a l . (1988) s u g g e s t s a more c o m p l i c a t e d p i c t u r e f o r e n a m e l i n . T h i s s t u d y d e m o n s t r a t e s a h e t e r o g e n e i t y w i t h i n e n a m e l i n s based on t h e i r g l y c o s y l a t i o n , e l e c t r o p h o r e t i c b e h a v i o u r and s t a i n a b i l i t y s u g g e s t i n g t h a t due t o p r o t e i n changes may be due t o a d e g r a d a t i o n o f e n a m e l i n d u r i n g t he m a t u r a t i o n o f e n a m e l . 3) M i n e r a l i z a t i o n / M a t u r a t i o n As p r e v i o u s l y s t a t e d enamel m i n e r a l i z a t i o n i s presumed t o o c c u r w i t h o u t t h e a i d o f t h e m a t r i x v e s i c l e b e c a u s e t h e y have no t been o b s e r v e d i n i n i t i a l enamel f o r m a t i o n ( B e r n a r d , 1 9 7 2 ) . I t has been s u g g e s t e d , based on EM a n a l y s i s , t h a t t h e enamel c r y s t a l s a r e d e r i v e d 20 f r om c r y s t a l s o f t h e f o r m i n g d e n t i n m a t r i x wh i ch i t b o r d e r s ( B e r n a r d , 1 9 7 2 ) . T h i s p o i n t i s e m p h a s i z e d because enamel c r y s t a l s a p p e a r i n t h e enamel m a t r i x soon a f t e r i t i s s e c r e t e d . A n a l y s i s o f t h e s e c r y s t a l s a t t h e DEJ w i t h c o n v e n t i o n a l EM t e c h n i q u e s i s d i f f i c u l t b e c a u s e o f a r t i f a c t s o f p r e p a r a t i o n and s e c t i o n i n g ( R o b i n s o n e t a l . , 1 9 8 1 ) . The q u e s t i o n c o n c e r n i n g t he o r i g i n o f enamel c r y s t a l s was a d d r e s s e d r e c e n t l y u s i n g SADF i m a g i n g , t h i s s t u d y s u g g e s t s a c o n t i n u i t y be tween d e n t i n and enamel c r y s t a l p o p u l a t i o n s ( A r s e n a u l t and R o b i n s o n , 1 9 8 9 ) . In a d d i t i o n t o f o r m i n g t he o r g a n i c m a t r i x , a m e l o b l a s t s a r e i n v o l v e d i n m i n e r a l i z a t i o n o f enamel by s e q u e s t e r i n g Ca2+, p r o v i d i n g an e n c l o s e d e n v i r o n m e n t , and i n c o n t r o l l i n g t h e o r g a n i z a t i o n o f c r y s t a l s w i t h i n t h e enamel m a t r i x . C a l c i u m i o n s e n t e r t h e enamel m a t r i x f rom b l o o d v e s s e l s i n t he s t a t u m i n t e r m e d i u m and t h e s t e l l a t e r e t i c u l u m t h r o u g h a m e l o b l a s t s (Munhoz and L e b l o n d , 1 9 7 4 ) . The mechanism o f Ca2+ r e l e a s e by a m e l o b l a s t s i n t o t he enamel m a t r i x may be by b o t h s e c r e t o r y g r a n u l e s and C a / A T P a s e a c t i v i t y o b s e r v e d a t t h e a p i c a l end o f a m e l o b l a s t s (Chen e t a l . , 1 9 8 6 ; S a s a k i and G a r a n t , 1 9 8 7 ) . A m e l o b l a s t s fo rm a t i g h l y bound l a y e r o f c e l l s , f r e e z e f r a c t u r e and c o n v e n t i o n a l EM have d e m o n s t r a t e d t i g h t j u n c t i o n s , desmosomes, and gap j u n c t i o n s between each c e l l ( S a s a k i e t a l . , 1 9 8 4 ) . T h i s l a y e r , l i k e t he o d o n t o b l a s t l a y e r , p r o v i d e s a l o c a l i z e d e n v i r o n m e n t o f c o n c e n t r a t e d CaP i o n s , bu t b e c a u s e o f c e l l movement and c e l l shape i t a l s o c o n t r o l s t h e o r g a n i z a t i o n o f enamel c r y s t a l s w i t h i n t h e m a t r i x ( O s b o r n , 1970 ; Warshawsky , 1 9 8 7 ) . M o r p h o l o g i c a l s t u d i e s have shown t h a t s e c r e t o r y a m e l o b l a s t s undergo d i f f e r e n t i a t i o n d u r i n g enamel f o r m a t i o n , w h i c h d i v i d e s i t i n t o 21 t h r e e s t a g e s . In t he f i r s t s t a g e a m e l o b l a s t s appea r a s low c o l u m n a r c e l l s wh i ch fo rm c r y s t a l s t h a t l i e p e r p e n d i c u l a r t o t h e D E J , t h i s i n i t i a l l a y e r o f enamel i s r e f e r r e d a s a p r i s m a t i c enamel ( L e b l o n d and Warshawsky , 1 9 7 9 ) . The a m e l o b l a s t s i n t he s e c o n d s t a g e become much t a l l e r and f o r m an a p i c a l p r o j e c t i o n - t he Tomes' p r o c e s s . Enamel m a t r i x i s s e c r e t e d f rom two a r e a s on t h e a p i c a l s u r f a c e o f t he a m e l o b l a s t : t h e Tomes ' p r o c e s s , wh i ch i s r e s p o n s i b l e f o r f o r m i n g r o d e n a m e l , and t h e a p i c a l membrane a t t he base o f Tomes' p r o c e s s w h i c h fo rms i n t e r r o d enamel ( J o h a n s e n , 1 9 6 4 ) . I n t e r r o d s u r r o u n d s t h e r o d enamel and i s c o n t i n u o u s w i t h t he i n i t i a l l a y e r o f e n a m e l . S c a n n i n g EM o f t h e s u r f a c e o f immature enamel w i t h t h e a m e l o b l a s t l a y e r removed r e v e a l s p r i s m s w i t h a c e n t r a l d e p r e s s i o n a r r a n g e d i n rows ( G r e e n b e r g e t a l . , 1 9 8 3 ; Warshawsky and L e b l o n d , 1 9 7 9 ) . C o m p a r a t i v e s t u d i e s have d e m o n s t r a t e d t he Tomes' p r o c e s s , and t h e r e f o r e t h e enamel p r i s m shape t o be s p e c i e s s p e c i f i c ( G r e e n b e r g e t a l . , 1 9 8 3 ; Warshawsky e t a l . , 1 9 8 1 ) . P r i s m s a r e made up o f i n t e r r o d enamel and i n l i f e t h e Tomes ' p r o c e s s e s f i l l t h e c e n t r a l d e p r e s s i o n , as t h e a m e l o b l a s t r e c e e d s w i t h enamel t h i c k e n i n g , r o d enamel o c c u p i e s t h e c e n t r a l s p a c e i n t he i n t e r r o d p r i s m . The re a r e two s e t s o f r o d s t h a t r un a t 60 - 90 d e g r e e s t o one a n o t h e r d e p e n d i n g upon where i n t h i s l a y e r t h e y a r e s a m p l e d ( J o d a i k i n e t a l . , 1 9 8 4 ) . S e r i a l s e c t i o n r e c o n s t r u c t i o n o f t h e s e s e t s o f r o d s show them t o r u n t h r o u g h t h i s e n t i r e l a y e r o f enamel (Warshawsky and S m i t h , 1 9 7 0 ) . Rod enamel i s composed o f b u n d l e s o f a l i g n e d a p a t i t e c r y s t a l s , i n t e r r o d enamel i s a l s o composed o f a p a t i t e , i t r u n s p e r p e n d i c u l a r t o t h e r o d s , bu t i s l e s s o r g a n i z e d ( G l i c k and E isenmann 1 9 7 3 ; Warshawsky e t a l . , 1 9 8 1 ) . • 22 In t he l a s t s t a g e o f enamel f o r m a t i o n t he a m e l o b l a s t l o s e s i t s Tomes ' p r o c e s s and d i m i n i s h e s i n h e i g h t . The a m e l o b l a s t f o rms an o u t e r a p r i s m a t i c l a y e r o f enamel w h i c h i s c o n t i n u o u s w i t h t h e f i r s t l a y e r and t he i n t e r r o d enamel o f t he s e c o n d l a y e r . These c e l l s a r e a l s o i n v o l v e d i n t h e m a t u r a t i o n o f t h e enamel m a t r i x ( L e b l o n d and w a r s h a w s k y , 1979 ; R e i t h and B o y d e , 1 9 8 1 ) . M a t u r a t i o n o f enamel i n v o l v e s t h e i n c r e a s e i n t he t h i c k n e s s and w i d t h o f enamel c r y s t a l s and t he r e d u c t i o n o f p r o t e i n c o n t e n t . T h i s p r o c e s s o c c u r s c o n t i n u a l l y t h r o u g h o u t t h e f o r m a t i o n o f e n a m e l , bu t d u r i n g t h e l a s t s t a g e i t i s a c c e l e r a t e d . The a m e l o b l a s t s a r e i n v o l v e d i n t h e c o n t i n u o u s m a t u r a t i o n by s e c r e t i n g p r o t e a s e s i n t o t he m a t r i x t o b reak down a m e l o g e n i n s , and t o a l e s s e r e x t e n t e n a m e l i n s ( L i m e b a c k , 1 9 8 7 ; O v e r a l l and L i m e b a c k , 1 9 8 8 ; S h i m i z u e t a l . , 1 9 7 9 ) , r e s o r b i n g t h e o r g a n i c m a t r i x and m a i n t a i n i n g t h e Ca c o n c e n t r a t i o n (Chen e t a l . , 1 9 8 6 ; K a l l e n b a c k , 1 9 7 6 ; Munhoz and L e b l o n d , 1 9 7 4 ; S a s a k i , 1 9 8 3 ) . A c c e l e r a t e d m a t u r a t i o n r a i s e s t h e m i n e r a l c o n t e n t f rom 70%, t o 96% due t o t he a c t i o n o f two c y c l i n g p o p u l a t i o n s o f m a t u r a t i v e a m e l o b l a s t s wh i ch r a p i d l y breakdown and remove enamel p r o t e i n s and i n c r e a s e Ca e n t r y i n t o t he m a t r i x (Crenshaw and T a k a n o , 1 9 8 2 ; R e i t h and B o y d e , 1 9 8 1 ) . D u r i n g m a t u r a t i o n t h e r e i s an i n c r e a s e i n t h e w i d t h and t h i c k n e s s o f enamel c r y s t a l s f rom 5 nm by 5 nm i n i n i t i a l enamel t o 60 nm by 30 nm i n t he most mature enamel ( N y l e n e t a l . , 1 9 6 3 ) . T h e r e i s , h o w e v e r , c o n s i d e r a b l e v a r i a t i o n i n enamel c r y s t a l measurement i n t h e l i t e r a t u r e b e c a u s e a g e , s p e c i e s and p o r t i o n o f enamel examined v a r y i n each s t u d y (Grove e t a l . , 1972 ; J e n s e n and M o l l e r , 1 9 4 8 ; D a c u l s i and K e r e b e l , 1 9 7 8 ) . I t has been s u g g e s t e d t h a t enamel c r y s t a l s become 23 v e r y l a r g e as a r e s u l t o f f u s i o n a s w e l l as c r y s t a l g row th ( D a c u l s i and K e r e b e l , 1 9 7 8 ) . The i n c r e a s e i n c r y s t a l s i z e due t o m a t u r a t i o n o b s e r v e d i n enamel i s u n l i k e any o t h e r mammalian m i n e r a l i z e d t i s s u e w h i c h seem t o have a r e s t r i c t e d c r y s t a l s i z e . 4) Enamel C r y s t a l s X - r a y d i f f r a c t i o n s t u d i e s have shown t h a t enamel c r y s t a l s , l i k e d e n t i n c r y s t a l s , have t h e u n i t c e l l d i m e n s i o n s o f a p a t i t e ( S e l v i g , 1 9 7 2 ) . I n i t i a l enamel c r y s t a l s , howeve r , have been d e s c r i b e d as b e i n g p o o r l y c r y s t a l l i n e w i t h e l e c t r o n d i f f r a c t i o n s t u d i e s . E l e c t r o n p robe m i c r o a n a l y s i s o f t h e s e c r y s t a l s shows them t o have a l o w e r m o l a r C a / P r a t i o t o o t h e r b i o l o g i c a l a p a t i t e ( L a n d i s e t a l . , 1 9 8 8 ) . W i t h i n c r e a s e d c r y s t a l g row th the e l e c t r o n d i f f r a c t i o n p a t t e r n becomes more d i s t i n c t , i n d i c a t i n g t h a t e l e c t r o n d i f f r a c t i o n p a t t e r n s o f i n i t i a l enamel may a p p e a r p o o r l y c r y s t a l l i n e due t o f e w e r , s m a l l e r c r y s t a l s a t t h i s s t a g e (Grove e t a l . , 1 9 7 2 ) . Based on measurements o f e x t r a c t e d e n a m e l , i t i s b e l i e v e d t h a t some enamel c r y s t a l s e x t e n d f rom the DEJ t o t h e s u r f a c e o f t h e enamel ( D a c u l s i e t a l . , 1984 ; S i m o n s , 1 9 7 2 ) . The shape o f t h e c r y s t a l s i s more d i f f i c u l t t o i n t e r p r e t due t o l i m i t a t i o n s i n t h e t r a n s m i s s i o n EM. Enamel c r y s t a l s were i n i t i a l l y i n t e r p r e t e d a s r i b b o n - l i k e h e x a g o n a l r o d s ( N y l e n e t a l . , 1 9 6 3 ) , b u t Warshawsky e t a l . (1987) d e t e r m i n e d t h a t the a p p e a r a n c e o f a h e x a g o n a l r i b b o n i s a r e s u l t o f o v e r p r o j e c t i o n o f a p a r a l l e l p i p e d shaped c r y s t a l . U s i n g s t e r e o - t i l t s and f r e e z e f r a c t u r e EM t h e y have d e m o n s t r a t e d t h e enamel c r y s t a l s t o be r e c t a n g u l a r r i b b o n s . 24 INTRODUCTION T o o t h m o r p h o g e n e s i s has been d e s c r i b e d as an e p i t h e l i a l - m e s e n c h y m a l o r g a n s y s t e m b e c a u s e p r o g r e s s i o n t h r o u g h i t s d e v e l o p m e n t a l s t a g e s o c c u r s a s a r e s u l t o f i n d u c t i v e i n t e r a c t i o n s be tween b o t h t he c e l l u l a r and t h e e x t r a c e l l u l a r components o f e p i t h e l i a l l y and mesenchyma l l y d e r i v e d t i s s u e s ( S l a v k i n e t a l . , 1 9 7 7 ; T h e s l e f f and H u r m e r i n t a , 1 9 8 1 ) . In v i t r o s t u d i e s o f t h i s p r o c e s s have shown t h a t t h e r e i s an i n t e r d e p e n d e n c y between t h e o r a l e p i t h e l i u m w h i c h d i f f e r e n t i a t e s i n t o enamel s e c r e t i n g a m e l o b l a s t s and t h e ectomesenchyme w h i c h d i f f e r e n t i a t e s i n t o d e n t i n f a r m i n g o d o n t o b l a s t s ( K o l l a r and B a i r d , 1 9 7 0 ; R u c h , 1984 ; T h e s l e f f and P r a t t , 1 9 8 0 ) . M o r p h o l o g i c a l s t u d i e s w h i c h d e m o n s t r a t e t he d e v e l o p m e n t a l and m i n e r a l i z a t i o n g r a d i e n t o f t o o t h m o r p h o g e n e s i s s u g g e s t t h a t a d i r e c t c o n t a c t between o d o n t o b l a s t s and a m e l o b l a s t s w h i c h o c c u r s j u s t p r i o r t o d e n t i n m i n e r a l i z a t i o n i s an i n d u c t i v e i n t e r a c t i o n wh i ch t r i g g e r s m i n e r a l i z a t i o n ( K a t c h b u r i a n and B u r g e s s , 1 9 7 7 ; 5 1 a v k i n and B r i n g a s , 1 9 7 6 ; S l a v k i n e t a l . , 1 9 7 7 ) . A f t e r t h i s i n t e r a c t i o n d e n t i n m i n e r a l i z a t i o n i s i n i t i a t e d i n t h e p r e - d e n t i n m a t r i x by m a t r i x v e s i c l e s s i m i l a r t o t h o s e f ound i n o t h e r m i n e r a l i z i n g c o l l a g e n o u s m a t r i c e s ( A r s e n a u l t 1 9 8 8 ; A r s e n u a l t e t a l . , 1988 ; B o n u c c i , 1 9 8 4 ; K a t c h b u r i a n , 1 9 7 3 ) . M i n e r a l i z a t i o n s p e a d s f rom t h e s e m a t r i x v e s i c l e s t h r o u g h t h e c o l l a g e n o u s m a t r i x t o w a r d s t he s u r f a c e w h i c h i s b o r d e r e d by a m e l o b l a s t s . Once m i n e r a l i z a t i o n r e a c h e s t h i s s u r f a c e enamel m a t r i x i s s e c r e t e d by a m e l o b l a s t s o n t o t h e d e n t i n . Enamel a p a t i t e c r y s t a l s can be o b s e r v e d w i t h i n t h i s m a t r i x soon a f t e r i t i s s e c r e t e d 25 and t h e y e x t e n d f rom the s u r f a c e o f t h e d e n t i n toward t he a m e l o b l a s t s ( B e r n a r d , 1972 ; E isenmann and G l i c k , 1 9 7 2 ; R e i t h , 1967 ; Warshawsky , 1 9 8 5 ) . T h i s s t a g e o f t o o t h m o r p h o g e n e s i s , where t h e enamel c r y s t a l s a r e i n i t i a l l y f o r m e d , i s no t w e l l u n d e r s t o o d . I t i s w e l l r e c o g n i z e d t h a t mammalian m i n e r a l i z a t i o n r e q u i r e s no t o n l y a s u p e r - s a t u r a t e d i o n i c c o n c e n t r a t i o n and a s t r u c t u r e d o r g a n i c m a t r i x , bu t a l s o t he p r e s e n c e o f m i n e r a l wh i ch w i l l a c t as an i n i t i a t i n g f a c t o r to m i n e r a l i z a t i o n . In d e n t i n and o t h e r m i n e r a l i z i n g c o l l a g e n o u s m a t r i c e s t h i s f a c t o r i s p r o v i d e d by m a t r i x v e s i c l e s . The i n i t i a t i o n o f enamel m i n e r a l i z a t i o n i s d i f f e r e n t f rom t h e s e o t h e r m i n e r a l i z i n g sys tems b e c a u s e i t l a c k s b o t h a c o l l a g e n o u s m a t r i x and m a t r i x v e s i c l e s . The q u e s t i o n o f how enamel c r y s t a l s a r e i n i t i a t e d d u r i n g enamel m i n e r a l i z a t i o n was f i r s t a d d r e s s e d by B e r n a r d ( 1 9 7 2 ) . He s u u g e s t e d t h a t enamel m i n e r a l i z a t i o n was a s e c o n d a r y m i n e r a l i z a t i o n p r o c e s s t h a t was dependen t upon d e n t i n m i n e r a l i z a t i o n . H i s m o r p h o l o g i c a l s t u d y o f t h e e a r l y e v e n t s o f enamel f o r m a t i o n i n t h e r a t m o l a r d e m o n s t r a t e d t h a t t h e r e was a c l o s e a s s o c i a t i o n be tween t he c r y s t a l s o f d e n t i n and e n a m e l , bu t he d i d no t show e v i d e n c e o f a d i r e c t c r y s t a l c o n t i n u i t y . The d e m o n s t r a t i o n o f a d i r e c t c o n t i n u i t y between d e n t i n and enamel wou ld be c o n s i s t e n t w i t h t h e h y p o t h e s i s f o r t h e r o l e o f d e n t i n as an i n i t i a t i n g f a c t o r f o r enamel m i n e r a l i z a t i o n . A l t h o u g h bo th enamel and d e n t i n have a p a t i t e as t h e i n o r g a n i c component o f t h e i r m a t r i c e s , t h e s i z e s and o r g a n i z a t i o n a l o f t h o s e c r y s t a l s a r e v e r y d i f f e r e n t . F a c t o r s w i t h i n t h e f i b r i l l a r and e x t r a f i b r i l l a r d e n t i n m a t r i x l i m i t t h e a p a t i t e t o a s m a l l c r y s t a l s i z e , bu t a l l o w f o r t h e i r random o r d e r i n g . In e n a m e l , t he o p p o s i t e i s t r u e a s v e r y l a r g e c r y s t a l s a r e h i g h l y o r g a n i z e d w i t h i n r o d and 26 i n t e r r e d d o m a i n s . T h i s v a r i e d o r d e r i n g i s r e f l e c t e d by t h e d i f f e r e n t c h e m i s t r y o f t h e o r g a n i c components o f t h e s e two m a t r i c e s ( B u t l e r , 1 9 8 4 a ; Te rm ine e t a l . , 1 9 7 9 ) . C r y s t a l s i z e can be used as a c r i t e r i o n t o d i s t i n g u i s h d e n t i n f rom enamel c r y s t a l s a t t he DEJ where t h e s e two g r o u p s o f c r y s t a l s a r e a s s o c i a t e d . The t e m p o r a l and s p a t i a l i n t e r r e l a t i o n s h i p o f d e n t i n and enamel f o r m a t i o n was s t u d i e d a t t h e EM l e v e l a l o n g t h e d e v e l o p m e n t a l g r a d i e n t o f t he DEJ i n t h e r a t m o l a r . M o r p h o l o g i c a l a s p e c t s o f t h e s e p r o c e s s e s were examined i n b o t h a q u e o u s l y and n o n - a q u e o u s l y p r e p a r e d t i s s u e s w i t h b r i g h t f i e l d and s e l e c t e d - a r e a dark f i e l d i m a g i n g t e c h n i q u e s . A r e v i e w o f t h e s t a g e s o f m i n e r a l i z a t i o n i n d e n t i n and enamel p r o v i d e d c o n f i r m a t i o n o f t h e e v e n t s r e p o r t e d by o t h e r a u t h o r s w h i c h i n c l u d e : t he i n i t i a t i o n o f d e n t i n m i n e r a l i z a t i o n by m a t r i x v e s i c l e s , t he f a c t t h a t d e n t i n m i n e r a l i z a t i o n p r e c e d e s enamel m i n e r a l i z a t i o n , and t h a t enamel c r y s t a l s a r e fo rmed on t he s u r f a c e o f d e n t i n and grow toward a m e l o b l a s t s ( B e r n a r d , 1 9 7 2 ; E i senmann and G l i c k , 1 9 7 2 ; R e i t h , 1 9 6 7 ) . A n o v e l v i e w o f changes t h a t o c c u r t o c o l l a g e n f i b r i l s a t t he edge o f p r e - d e n t i n j u s t p r i o r t o m i n e r a l i z a t i o n s u g g e s t s t h a t t h e s e p r e - d e n t i n c o l l a g e n f i b r i l s have a s p e c i f i c r o l e i n enamel m i n e r a l i z a t i o n . The p r e s e n c e o f an enamel s h e a t h w h i c h b o r d e r s t he a p a t i t e c r y s t a l s i n enamel i s r e v i e w e d w i t h s p e c i f i c i n t e r e s t i n t he s h e a t h ' s r e l a t i o n s h i p t o t he s u r f a c e o f d e n t i n a t one end and t h e a m e l o b l a s t c e l l membrane a t t h e o t h e r . The a p a t i t e c r y s t a l o f d e n t i n i s i n v e s t i g a t e d w i t h s e l e c t e d - a r e a d a r k f i e l d t o d e t e r m i n e i f t h e r e i s an o r g a n i z a t i o n w i t h i n i t . C r y s t a l c o n t i n u i t y between a p a t i t e o f d e n t i n and enamel a t t he DEJ i n b o t h e a r l y and more mature s t a g e s o f t o o t h d e v e l o p m e n t . The i m p l i c a t i o n s o f 27 t h e s e r e s u l t s a r e d i s c u s s e d i n l i g h t o f a w o r k i n g model o f t h e s p a t i a l r e l a t i o n s h i p s between d e n t i n and d e v e l o p i n g e n a m e l . From t h i s r e s e a r c h i t a p p e a r s t h a t enamel a p a t i t e c r y s t a l s f o rm a s a s e c o n d a r y m i n e r a l i z a t i o n p r o c e s s , a s t h e r e i s a i n t i m a t e s p a t i a l r e l a t i o n s h i p be tween b o t h t h e o r g a n i c and i n o r g a n i c components o f d e n t i n and e n a m e l . 28 MATERIALS AMD METHODS 1) A n i m a l s F o r each e x p e r i m e n t , m o l a r s were e x t r a c t e d f rom t h e m a n d i b l e s o f 3 t o 7 d a y - o l d , male Spague -Daw ley r a t s . T h i s age r a n g e was s e l e c t e d b e c a u s e a l l d e v e l o p m e n t a l s t a g e s o f enamel and d e n t i n a r e p r e s e n t . M a n d i b u l a r m o l a r s were used b e c a u s e t h e e a r l y d e v e l o p m e n t a l s e q u e n c e s a r e more e a s i l y i d e n t i f i e d g r o s s l y t h a n i n t he i n c i s o r ; t h i s was most i m p o r t a n t f o r t h e s l a m f r e e z i n g f i x a t i o n . 2) Me thods o f F i x a t i o n f o r TEM: a) C o n v e n t i o n a l Spec imen P r e p a r a t i o n i ) Immers ion F i x a t i o n : R a t s were k i l l e d by d e c a p i t a t i o n and t h e i r m a n d i b l e s were e x c i s e d , s p l i t a t t h e i r s y m p h y s i s and immersed i n 2 .3% g l u t a r a l d e h y d e i n 0 . 0 5 M sod ium c a c o d y l a t e , b u f f e r e d t o 300 mOsm. w i t h 5% s u c r o s e and a d j u s t e d t o pH 7 . 3 , a t 4°C f o r 2 h r s . T i s s u e s were washed t w i c e f o r 15 min i n sod ium c a c o d y l a t e b u f f e r a t room t e m p e r a t u r e , and t h e n immersed i n a 1% b u f f e r e d s o l u t i o n o f osmium t e t r o x i d e f o r 1 .5 h r s . The t i s s u e was washed a g a i n a s above and t h e n d e h y d r a t e d i n a g raded s e r i e s o f m e t h a n o l ( 5 0 , 7 0 , 9 0 , 1 0 0 % ) f o r 15 min e a c h , and t h r e e changes a t 100% b e f o r e b e i n g t r a n s f e r r e d t o a c e t o n e ( t h r e e changes a s w e l l ) . i i ) P e r f u s i o n F i x a t i o n : R a t s a n e s t h e t i s e d w i t h sod ium p e n t a b a r b i t o l were f i r s t p e r f u s e d i n t r a c a r d i a l l y w i t h R i n g e r ' s s a l i n e w h i c h c o n t a i n e d an a n t i - c o a g u l a n t (0 .025% h e p a r i n ) , a musc le r e l a x a n t (0 .1% p r o c a i n e ) and was a d j u s t e d t o 300 mOsm w i t h 5% s u c r o s e and pH 7 . 3 . T h i s s a l i n e , l i k e a l l o t h e r p e r f u s a t e s u s e d , was warmed t o 37°C 29 and i t s f l o w t h r o u g h a c a n n u l a was a d j u s t e d t o 40 m l s / m i n w i t h a p r e s s u r e o f 100 mmHg t o a p p r o x i m a t e p h y s i o l o g i c a l c o n d i t i o n s . S a l i n e was p e r f u s e d u n t i l a l l o f t he b l o o d was c l e a r e d , as o b s e r v e d by c l e a r s a l i n e f l o w i n g f rom the c u t e x t e r n a l j u g u l a r v e i n s . A f t e r c l e a r i n g t h e p e r f u s a t e was s w i t c h e d t o 2.3% g l u t a r a l d e h y d e i n 0 . 0 5 Fl sod ium c a c o d y l a t e b u f f e r e d t o pH 7 . 3 and 300 mOsm f o r 20 m i n . F l a n d i b l e s f r om e a c h r a t were e x c i s e d , s p l i t a t t h e s y m p h y s i s and immersed i n t h e above f i x a t i v e c o o l e d t o 4 a C f o r an a d d i t i o n a l h o u r . D u r i n g t h i s f i x a t i o n t h e h a l f m a n d i b l e s were d i v i d e d f u r t h e r a t t he g r o o v e d e l i n e a t i n g t he d i v i s i o n between t h e bony c a v i t i e s c o n t a i n i n g t h e m o l a r s and t he i n c i s o r . S e c t i o n s o f t h e m a n d i b l e c o n t a i n i n g two m o l a r s were washed i n the sod ium c a c o d y l a t e b u f f e r , p r e p a r e d a s m e n t i o n e d a b o v e , two t i m e s f o r 15 min t h e n immersed i n a b u f f e r e d 1% s o l u t i o n o f osmium t e t r o x i d e f o r 1 .5 h r s . The t i s s u e was t h e n washed and d e h y d r a t e d a s i n t he i m m e r s i o n f i x a t i o n . b) C r y o g e n i c Spec imen P r e p a r a t i o n E x c i s e d m o l a r s were s l i c e d i n h a l f and p l a c e d i n on f i l t e r p a p e r w i t h t he c u t s u r f a c e f a c i n g u p . T h e s e were i n t u r n p l a c e d on a t e f l o n d i s c and s l am f r o z e n on a h i g h p u r i t y c o p p e r b l o c k c o o l e d t o l i q u i d h e l i u m t e m p e r a t u r e s ( - 2 7 0 ° C) ( A r s e n a u l t e t a l . , 1 9 8 8 ) . The d i s c w i t h t h e f r o z e n spec imen a t t a c h e d was p l a c e d i n l i q u i d n i t r o g e n u n t i l t r a n s f e r t o p r e - c o o l e d ( - 8 5 ° C ) g l a s s v i a l s w i t h 0.5% g l u t a r a l d e h y d e i n 100% m e t h a n o l . In t h e s e v i a l s t i s s u e s were f r e e z e - s u b s t i t u t e d f o r 48 h r s a t - 8 5 C t h e n t h e t e m p e r a t u r e was g r a d u a l l y r a i s e d t o room t e m p e r a t u r e o v e r a p e r i o d o f 2 . 5 h r s . A f t e r t h r e e washes o f 100% methano l and t h r e e o f 100% a c e t o n e t he s p e c i m e n 30 were i n f i l t r a t e d and embedded a s d e s c r i b e d b e l o w . c ) A i r D r y i n g Spec imen P r e p a r a t i o n E x c i s e d m o l a r s were a l l o w e d t o a i r d r y f o r 3 w e e k s . The m o l a r s were t h e n washed i n a c e t o n e and embedded i n S p u r r r e s i n a s d e s c r i b e d b e l o w . 3) Embeddment and M i c ro tomy A f t e r d e h y d r a t i o n i n m e t h a n o l and a c e t o n e t h e s p e c i m e n s p r e p a r e d f o r TEM were i n f i l t a t e d w i t h S p u r r r e s i n i n a c e t o n e ( 1 : 3 , 1 : 1 , 3 : 1 , 1 0 0 % ) . I n f i l t r a t i o n t i m e s were 4 - 6 h r s f o r each o f t h e f i r s t 3 s t e p s and o v e r n i g h t f o r 100% r e s i n . The n e x t day t h e s p e c i m e n i n 100% r e s i n were p l a c e d f o r 1 5 - 3 0 min i n a vacuum d e s i c c a t o r t o remove a i r and t h e n p l a c e d i n an oven a t 60 C f o r 8 -12 h r s . T h i c k s e c t i o n s ( 0 . 5 - 1 urn) were c u t w i t h a d iamond k n i f e on a P o r t e r B lum u l t r a m i c r o t o m e , p l a c e on g l a s s s l i d e s and s t a i n e d w i t h 1% aqueous t o l u i d i n e b l u e t o e s t a b l i s h o r i e n t a t i o n . T h i n s e c t i o n s ( s i l v e r , 5 0 - 6 0 nm) were t h e n c u t and p i c k e d up on f o r m v a r - c o a t e d c o p p e r g r i d s and were e i t h e r s t a i n e d (as d e s c r i b e d be low) o r l e f t u n s t a i n e d . C r y o p r e p a r e d s p e c i m e n s were embedded and o r i e n t e d a s d e s c r i b e d a b o v e ; h o w e v e r , u l t r a t h i n s e c t i o n s , ( t r a n s p a r e n t , <50 nm) needed f o r a p a t i t e a n a l y s i s w i t h s e l e c t e d - a r e a da rk f i e l d i m a g i n g , were c u t w i t h a d iamond k n i f e and were p i c k e d up on u n c o a t e d c o p p e r g r i d s . F i v e t o t e n s e r i a l s e c t i o n s ( g r e y / s i l v e r , 6 0 - 9 0 nm) were c u t i n d i f f e r e n t b l o c k s and were o b s e r v e d u n s t a i n e d on f o r m v a r c o a t e d g r i d s . 31 4) S t a i n i n g The m i n e r a l i z e d components of the tooth have inherent e l e c t r o n density that can be v i s u a l i z e d unstained by the e l e c t r o n microscope. The nonmineralized components and c e l l s ; however, are not s u f f i c i e n t l y dense to provide u l t r a s t r u c t u r a l d e f i n i t i o n , so heavy metal s t a i n s are used to enhance c o n t r a s t . Conventionally prepared specimens on copper g r i d s were suspended i n 1% uranyl acetate i n double d i s t i l l e d water (adjusted to pH 4.2) f o r 15 minutes, washed i n double d i s t i l l e d water f o r 30-60 seconds, s t a i n e d i n Reynold's Lead f o r 10 minutes. Specimens on formvar coated copper g r i d s were demineralized with ethylenediaminetetra-acetic a c i d (EDTA) (Warshawsky and Moore 1967), washed, then stained with \% phosphotungstic a c i d i n double d i s t i l l e d water (pH 3.2) f o r 25 minutes, washed, then s t a i n e d with 1% uranyl acetate i n double d i s t i l l e d water (pH 4.2) f o r 10 minutes. These were then washed i n d i s t i l l e d water before being a i r d r i e d and viewed with the TEM. Some co n v e n t i o n a l l y prepared t i s s u e were not demineralized with EDTA, but were s t a i n e d with phosphotungstic a c i d and uranyl acetate. 5) Imaging Techniques B r i g h t f i e l d (BF) TEM was used to image both s t a i n e d and unstained specimens. In b r i g h t f i e l d EM images are formed by the unscattered e l e c t r o n s which pass through the specimen and are c o l l e c t e d by the underlying o b j e c t i v e aperture. In c o n t r a s t , i n a SADF image only the d i f f r a c t e d e l e c t r o n s are c o l l e c t e d by the o b j e c t i v e aperture. With c r y s t a l l i n e m a t e r i a l s , l i k e a p a t i t e , 32 s p e c i f i c d i f f r a c t i o n p a t t e r n s a r e fo rmed when e l e c t r o n s a r e t r a n s m i t t e d t h r o u g h t h e s p e c i m e n . F o r a p a t i t e t h e s e d i f f r a c t e d e l e c t r o n s g i v e maxima ( d - s p a c i n g s ) w h i c h can be c o l l e c t e d by t h e o b j e c t i v e a p e r t u r e and i n t u r n i m a g e d . I n t h i s s t u d y e l e c t r o n r e f l e c t i o n s r e p r e s e n t i n g the a b - [ 1 0 2 , 2 1 0 , 2 1 1 , 1 1 2 , 3 0 0 , 2 0 2 , and 301 d - s p a c i n g s ] , c - [002 d - s p a c i n g ] and comb ined a b c - a x e s o f a p a t i t e were imaged by SADF. The a b - and c - a x i a l r e f l e c t i o n s were s e l e c t e d u s i n g a 4 pn o b j e c t i v e a p e r t u r e , w h i l e t h e comb ined a b c - a x e s were imaged u s i n g a 10 urn a p e r t u r e . B o t h t h e P h i l i p s 300 and 301 TEMs were u s e d i n b r i g h t f i e l d a t a c c e l e r a t i n g v o l t a g e s o f 60 and 80 kV f o r s t a i n e d s p e c i m e n s . The P h i l i p s 300 TEM was a l s o used a t 80 kU f o r b r i g h t and da rk f i e l d images o f u n s t a i n e d s p e c i m e n s . 33 RESULTS L o n g i t u d i n a l p l a n e s o f s e c t i o n s t h r o u g h t h e t o o t h a t t h i s e a r l y s t a g e o f deve lopmen t r e v e a l m o n o l a y e r s o f o d o n t o b l a s t s and a m e l o b l a s t s s e c r e t i n g t h e i r r e s p e c t i v e e x t r a c e l l u l a r m a t r i c e s d e n t i n and enamel ( F i g . 1 ) . T h i s LM image o f a 0 . 5 urn t h i c k p l a s t i c s e c t i o n p r o v i d e s an o v e r v i e w o f o d o n t o g e n e s i s and s e r v e s t o f o c u s a t t e n t i o n on t h e DEJ w h i c h i s t he s i t e o f p o s s i b l e i n t e r a c t i o n between t h e o r g a n i c components and a p a t i t e c r y s t a l s o f t h e s e two m a t r i c e s . The o d o n t o b l a s t s and a m e l o b l a s t s a r e s e p a r a t e d by e x t r a c e l l u l a r m a t r i c e s o f d e n t i n and e n a m e l . The e n c l o s e d a r e a s ( b , c , d , e ) o f t h i s image c o r r e s p o n d t o r e p r e s e n t a t i v e a r e a s o b s e r v e d a t t h e EM l e v e l a s shown i n F i g u r e s 1 b - e r e s p e c t i v e l y . F i g u r e 1b shows t h e p r e - d e n t i n m a t r i x s e p a r a t e d f rom an a m e l o b l a s t by a BM, t h i s m a t r i x c o n t a i n s c o l l a g e n f i b r i l s , g round s u b s t a n c e , m a t r i x v e s i c l e s , and o d o n t o b l a s t p r o c e s s e s . C o l l a g e n f i b r i l s appea r t o nar row a t t h e i r t e r m i n a l p o r t i o n s w h i c h a s s o c i a t e w i t h t h e BM. M a t r i x v e s i c l e s have d i f f e r e n t s t a i n i n g p r o p e r t i e s and shapes depend ing upon t h e i r c o n t e n t s and t h e y a r e u s u a l l y f o u n d i n c l o s e p r o x i m i t y t o t he BM ( S l a v k i n e t a l . , 1 9 7 2 ) . The a m e l o b l a s t s w h i c h r e s t on t he BM have r i b o s o m e s and v e s i c l e s i n t h e i r a p i c a l c y t o p l a s m . F i g u r e 1c d e m o n s t r a t e s t h e changes t h a t o c c u r i n t h i s r e g i o n a s p r e d e n t i n m i n e r a l i z e s : t h e BM has been b r o k e n down and remnan ts o f i t can be o b s e r v e d between o u t f o l d i n g s o f t h e a m e l o b l a s t a p i c a l membrane, and m i n e r a l d e p o s i t i o n s p r e a d s f rom i s o l a t e d l o c i w i t h i n p r e - d e n t i n a l o n g c o l l a g e n f i b r i l s . U n f o r t u n a t e l y due t o r o u t i n e s t a i n i n g p r o c e d u r e s w h i c h r e s u l t i n t h e d e p l e t i o n o f a l l d e t e c t a b l e c a l c i u m p h o s p h a t e and a p a t i t e ; t he p r e v i o u s l y 34 m i n e r a l i z e d m a t r i x can o n l y be o b s e r v e d due t o t he s t r u c t u r a l m o d i f i c a t i o n o f t h e m a t r i x d u r i n g m i n e r a l i z a t i o n w h i c h i n c o r p o r a t e s heavy m e t a l s d u r i n g t he s t a i n i n g p r o c e d u r e ( A r s e n a u l t and H u n z i k e r , 1 9 8 8 ) . In t h i s m i c r o g r a p h , m i n e r a l i z a t i o n a d v a n c e s t oward t he a p i c a l membrane o f a m e l o b l a s t s a l o n g c o l l a g e n f i b r i l s w h i c h c o n t r i b u t e t o t h e uneven a p p e a r a n c e o f t he s u r f a c e o f d e n t i n a t t h i s s t a g e . The more i n t e n s e s t a i n i n g o f t he d e n t i n s u r f a c e a p p e a r s t o be a n a l a g o u s t o t h e l a m i n a l i m i t a n s d e m o n s t r a t e d i n s t a i n d e p o s i t s o f bone and c a r t i l a g e by S c h e r f t ( 1 9 7 2 ) . He has s u g g e s t e d t h a t t h i s e l e c r o n - d e n s e l a y e r t h a t b o r d e r s m i n e r a l i z e d r e g i o n s i s f o rmed as a r e s u l t o f a c c r e t i o n o f o r g a n i c m a t e r i a l between and a g a i n s t a p a t i t e a t t h e m i n e r a l s u r f a c e , o r by a p r o c e s s o f a d s o r p t i o n . The a p i c a l o u t f o l d i n g s o f t h e a m e l o b l a s t appea r e x t e n d e d t o w a r d t h e p r e - d e n t i n and a r e i n c l o s e p r o x i m i t y t o c o l l a g e n f i b r i l s and an o d o n t o b l a s t p r o c e s s . The c l o s e p r o x i m i t y o f an o d o n t o b l a s t p r o c e s s t o an a m e l o b l a s t g i v e s e v i d e n c e f o r a p o s s i b l e m e s e n c h y m a l / e p i t h e l i a l c e l l - t o - c e l l c o m m u n i c a t i o n t r i g g e r i n g t h e s e c r e t i o n o f t h e enamel m a t r i x ( S l a v k i n and B i n g a s , 1976 ; K a t c h b u r i a n and B u r g e s s , 1 9 7 7 ) . In t he a m e l o b l a s t , e l e c r o n - d e n s e s e c e t o r y p r o d u c t i s s t o r e d i n c y t o p l a s m i c v e s i c l e s n e a r t h e a p i c a l c e l l membrane u n t i l m i n e r a l i z a t i o n r e a c h e s t he edge o f d e n t i n . A f t e r m i n e r a l i z a t i o n r e a c h e s t h e s u r f a c e o f d e n t i n t h e enamel m a t r i x i s e x o c y t o s e d o n t o d e n t i n ( F i g . 1 d ) . The i n i t i a l enamel m a t r i x has been d e s c r i b e d a s s t i p p l e d m a t e r i a l because o f i t s p a r t i c u l a t e n a t u r e ( R e i t h , 1 9 6 8 ) . Enamel c r y s t a l s a r e e l e c t r o n - d e n s e n e e d l e - l i k e s t r u c t u r e s w h i c h e x t e n d f rom t h e s u r f a c e o f d e n t i n t h r o u g h t h e s t i p p l e d enamel m a t r i x t o w a r d a m e l o b l a s t s . I n i t i a l l y t h e s e c r y s t a l s a r e i n d i s t i n g u i s h a b l e f r om 35 FIGURE 1 - OVERVIEW OF THE FORMATION OF THE DENTINO-ENAMEL JUNCTION (a) A 0.5 urn t h i c k s e c t i o n s t a i n e d with t o l u i d i n e blue shows f i v e l a y e r s of c e l l s . The s t e l l a t e r e t i c u l u m (SR), the stratum intermedium ( S i ) , and the ameloblast l a y e r (A) make up the supportive and s e c r e t o r y c e l l s of enamel. The dental pulp c e l l s (DP), and the odontoblast l a y e r (0) are the supportive and secretory c e l l s of de n t i n . Enclosed areas of i n t e r e s t are shown at higher m a g n i f i c a t i o n below. Bar = 20 urn; Mag = 800x. (b) A p o r t i o n of an ameloblast (A) i s separated from the predentin matrix (PD) by a t h i n basal lamina (BL). Within the predentin matrix, and close to the basal lamina are matrix v e s i c l e s (MV), a c e l l process of an odontoblast (OP), and numerous randomly arranged c o l l a g e n f i b r i l s . (c) During dentin (D) formation, m i n e r a l i z a t i o n occurs between and along c o l l a g e n f i b r i l s (small arrowheads). Here m i n e r a l i z a t i o n appears to be progressing toward the ameloblast (A) which has penetrated the basal lamina. An odontoblast process can be observed i n c l o s e proximity to the a p i c a l membrane of an ameloblast. Within ameloblasts enamel products are stored i n cytoplasmic v e s i c l e s ( l a r g e arrowheads). (d) The enamel matrix i s released as a s t i p p l e d m a t e r i a l (SM) by the ameloblast (A) onto the surface of dentin (D), forming a l a y e r 36 o f enamel ( E ) . The a m e l o b l a s t i s a l s o i n v o l v e d i n r e s o r p t i o n , i n d i c a t e d by t h e p r e s e n c e o f a p i n o c y t o t i c - l i k e v e s i c l e ( a r r o w h e a d ) . (e ) As more enamel m a t r i x i s r e l e a s e d by t h e a m e l o b l a s t t h e enamel l a y e r (E) t h i c k e n s . B a r = 500 nm; Mag = 3 6 , 0 0 0 x . [ b - e were c o n v e n t i o n a l l y p r e p a r e d and s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e ( U A / L C ) ] i 37 38 d e n t i n c r y s t a l s , e x c e p t t h a t t h e y a r e o b s e r v e d w i t h i n t h e enamel m a t r i x . F u r t h e r a l o n g t he DEJ more m a t r i x m a t e r i a l has been r e l e a s e d by t h e a m e l o b l a s t and so the enamel l a y e r i s t h i c k e r ; h e r e t h e enamel c r y s t a l s a p p e a r t o be i n c l o s e p r o x i m i t y t o t h e a p i c a l c e l l membrane ( F i g . 1 e ) . E l e c t r o n d e n s i t i e s r e s e m b l i n g a p a t i t e c r y s t a l s a r e f i r s t o b s e r v e d w i t h i n m a t r i x v e s i c l e s i n p r e - d e n t i n ( K a t c h b u r i a n , 1 9 7 3 ) . F i g u r e 2b shows a m a t r i x v e s i c l e t h a t i s c o n t a i n e d w i t h i n t h e t h i c k n e s s o f a s e c t i o n ( 6 0 - 9 0 nm t h i c k ) a s t h i s i s t he s e c o n d s e c t i o n i n a s e r i e s o f t h r e e . ( F i g s . 2 a - c ) . These m i c r o g r a p h s a l s o d e m o n s r a t e t h e e x t e n s i v e r e t i c u l a r f i b r i l s a s s o c i a t e d w i t h t h e BM and w i t h t h e random ly a r r a n g e d c o l l a g e n f i b e r s o f p r e - d e n t i n . An u n s t a i n e d s e c t i o n o f p r e - d e n t i n shows t h e r a d i a l a r r a n g e m e n t o f m i n e r a l d e p o s i t i o n i n t h r e e m i n e r a l i z i n g l o c i ( F i g . 2 d ) . The s m a l l e s t l o c u s may be a m a t r i x v e s i c l e , bu t w i t h o u t s t a i n i n g t o s e e a l i m i t i n g membrane i t i s d i f f i c u l t t o i d e n t i f y i t f r om a c r o s s - s e c t i o n o f a m i n e r a l i z e d c o l l a g e n f i b r i l . The e l e c t r o n - d e n s e n e e d l e - l i k e s t r u c t u r e s a r e d e n t i n c r y s t a l s and t he e l e c t r o n - d e n s i t y s u r r o u n d i n g t h e s e c r y s t a l s may be due t o an i n c r e a s e d c o n c e n t r a t i o n o f c a l c i u m and p h o s p h a t e i o n s . M i n e r a l i z a t i o n i s i n i t i a t e d w i t h i n m a t r i x v e s i c l e s and t h e n r a d i a t e s t h r o u g h t h e a f i b r i l l a r r e g i o n s t o c o l l a g e n f i b r i l s ( K a t c h b u r i a n , 1 9 7 3 ) . Somet imes i f t he v e s i c l e i s v e r y c l o s e t o a c o l l a g e n f i b r i l , m i n e r a l i z a t i o n w i l l f o l l o w t h e c o l l a g e n f i b r i l b e f o r e r a d i a t i n g i n t o t h e a f i b r i l l a r r e g i o n ( F i g . 2 e ) . T h i s p r e f e r e n t i a l m i n e r a l i z a t i o n o f c o l l a g e n f i b r i l s , where m i n e r a l i z a t i o n p r o c e e d s a l o n g c o l l a g e n f i b r i l s more r a p i d l y t h a n t h r o u g h a f i b r i l l a r m a t r i x r e g i o n s , has been r e p o r t e d by many a u t h o r s i n d e n t i n and o t h e r c o l l a g e n o u s t i s s u e m i n e r a l i z a t i o n 39 ( A r s e n a u l t , 1 9 8 8 ; G l i m c h e r , 1979 ; H o h l i n g e t a l . , 1976 ; K a t c h b u r i a n , 1 9 7 3 ) . T h i s phenomena i s no t o n l y o b s e r v e d i n t h e f o r m a t i o n o f m a n t l e d e n t i n ( f i r s t f o r m e d ) , bu t a l s o i s s e e n i n t he c i r c u m p u p a l d e n t i n ( s u b s e q u e n t l y fo rmed) a t t he p r e - d e n t i n d e n t i n b o r d e r ( F i g s . 2 f and g ) . A c o l l e c t i o n o f c o l l a g e n f i b r i l s a t t h e p r e - d e n t i n / d e n t i n j u n c t i o n d e m o n s t r a t e s t he changes t h a t o c c u r upon m i n e r a l i z a t i o n ( F i g . 2 g ) . In t h e d e n t i n , t h e s e f i b r i l s a p p e a r l a r g e r and have an a x i a l b a n d i n g p a t t e r n wh ich i s more d i f f u s e t h a n t h o s e i n p r e - d e n t i n . F i g u r e 2g i s a l o w e r m a g n i f i c a t i o n o f a r e g i o n s i m i l a r t o F i g . 2 f s h o w i n g t h e p r e - d e n t i n / d e n t i n b o r d e r w i t h o u t s t a i n i n g . The dense a r e a i n t h e c e n t r e o f t he d e n t i n r e s u l t s f r o m t h e m i n e r a l i z a t o n o f t h e c l o s e l y p a c k e d c o l l a g e n f i b r i l s i n a von K o r f f f i b r e o r i e n t e d p e r p e n d i c u l a r t o t he p r e - d e n t i n / d e n t i n b o r d e r . The von K o r f f f i b r e s a p p e a r t o be r e s p o n s i b l e f o r e x t e n s i o n s o f m i n e r a l i z a t i o n i n t o p r e - d e n t i n ( B e r n a r d , 1 9 7 2 ) , s u g g e s t i n g t h a t m i n e r a l i z a t i o n grows more r a p i d l y a l o n g t he o r g a n i z e d c o l l a g e n o u s m a t r i x t h a n t h r o u g h t h e l e s s / s t r u c t u r e d a f i b r i l l a r m a t r i x . F i g u r e s 3a and b a r e m i c r o g r a p h s o f t h e p r e - d e n t i n / a m e l o b l a s t j u n c t i o n a t a s t a g e j u s t p r i o r t o t h e s e c r e t i o n o f enamel m a t r i x ( F i g . 1 c ) . The BM has been removed and p r o j e c t i o n s f r om a m e l o b l a s t s e x t e n d i n t o p r e - d e n t i n , between t h e s e p r o j e c t i o n s j a v e l i n p o i n t e d c o l l a g e n f i b r i l s can be o b s e r v e d . P i n o c y t o t i c - l i k e v e s i c l e s i n t he a m e l o b l a s t n e a r i t s a p i c a l membrane s u g g e s t t h a t i t i s i n v o l v e d i n r e s o r p t i o n o f t h e BM a s p o s t u l a t e d by R e i t h ( 1 9 6 7 ) . A t a l a t e r d e v e l o p m e n t a l s t a g e , a f t e r a p r o l o n g e d d e m i n e r a l i z a t i o n and s t a i n i n g , c o l l a g e n f i b r i l s a r e d i s c e r n a b l e f rom the r e s t o f t he m i n e r a l i z e d m a t r i x ( F i g . 3 c ) . U n l i k e t h e c o l l a g e n f i b r i l s o f F i g s . 3a and b , t h e s e f i b r i l s have a l a r g e r 40 FIGURE 2 - IN IT IATION AND SUBSEQUENT MINERALIZATION OF DENTIN ( a - c ) A s e r i e s o f m i c r o g r a p h s o f s e r i a l s e c t i o n s , s h o w i n g a m a t r i x v e s i c l e (MV) t o be w i t h i n t h e t h i c k n e s s o f t h e s e c o n d s e c t i o n . An a m e l o b l a s t (A) r e s t s on a b a s a l l a m i n a ( B L ) . C o l l a g e n f i b r i l s ( s m a l l a r r o w h e a d s ) i n t he p r e d e n t i n a r e randomly a r r a n g e d o b s e r v e d h e r e c u t i n t r a n s v e r s e and c r o s s s e c t i o n (a) t o be d i s t i n c t f r o m t h e o d o n t o b l a s t p r o c e s s ( O P ) . B a r = 200 nm; Mag = 5 2 , 0 0 0 x . [ c o n v e n t i o n a l l y p r e p a r e d and s t a i n e d w i t h U A / L C ] (d) An u n s t a i n e d e l e c t r o n m i c r o g r a p h o f c o n v e n t i o n a l l y p r e p a r e d p r e d e n t i n shows t h r e e p r o g r e s s i v e s t a g e s o f m i n e r a l i z a t i o n . The a r r o w h e a d p o i n t s t o an e l e c t r o n - d e n s e r e g i o n s u r r o u n d i n g a s i n g l e a p a t i t e c r y s t a l , p r o b a b l y a m a t r i x v e s i c l e . The o t h e r two e l e c t r o n - d e n s e a r e a s c o n t a i n more m i n e r a l , w h i c h a p p e a r s t o r a d i a t e f rom a c e n t r a l p o i n t . Two a m e l o b l a s t c e l l u l a r p r o j e c t i o n s c a n be o b s e r v e d above t he p r e d e n t i n . B a r = 200 nm; Mag = 7 8 , 0 0 0 x . (e ) M i n e r a l i z a t i o n i n d e n t i n p r o g r e s s e s f rom m a t r i x v e s i c l e s ( a r r o w h e a d s ) t o c o l l a g e n f i b r i l s , t h r o u g h t h e a f i b r i l l a r m a t r i x , t h e n a l o n g t h e randomly a r r a n g e d c o l l a g e n f i b r i l s . B a r = 400 nm; Mag = 4 7 , 0 0 0 x . [ c o n v e n t i o n a l l y p r e p a r e d and s t a i n e d w i t h UA /LC ] ( f ) O r g a n i z e d c o l l a g e n f i b r i l s o f a von K o r f f f i b r e d e m o n s t r a t e t h e p r e d e n t i n ( P D ) / d e n t i n (D) b o r d e r . The d i s t i n c t p e r i o d i c b a n d i n g o f t he p r e - d e n t i n p o r t i o n o f t h e s e c o l l a g e n f i b r i l s 41 a p p e a r t o be r e d u c e d i n t h e d e n t i n p o r t i o n . B a r = 1 5 0 nm; Mag = 7 7 , 0 0 0 x . [ c o n v e n t i o n a l l y p r e p a r e d and s t a i n e d w i t h P h o s p h o t u n g s t i c A c i d ( P T A ) / U A ] (g ) A l o w e r m a g n i f i c a t i o n o f an u n s t a i n e d s e c t i o n i n a s i m i l a r a r e a t o F i g u r e 2 f . A von K o r f f f i b r e (VK) i s more d e n s e l y m i n e r a l i z e d t h a n t h e s u r r o u n d i n g d e n t i n ( D ) . B a r = 200 nm; Mag = 3 1 , 0 0 0 x . [ c o n v e n t i o a l l y p r e p a r e d and no t s t a i n e d ] 42 43 d i a m e t e r (1 t o 2 t i m e s i n t he main p o r t i o n and 5 t o 12 t i m e s f o r t he t e r m i n a l p o r t i o n ) , and a r e d u c e d a x i a l b a n d i n g i n t h e i r t e r m i n a l p o r t i o n w h i c h a p p e a r s t o be f r a y e d r a t h e r t h a n p o i n t e d . T h i n e l e c t r o n - l u c e n t l i n e s i n t h e dense enamel m a t r i x a r e s p a c e s o c c u p i e d by ename l c r y s t a l s p r i o r t o d e m i n e r a l i z a t i o n , t h e y a r e f o u n d t o be a s s o c i a t e d w i t h t he t e r m i n a l , f r a y e d p o r t i o n o f d e n t i n c o l l a g e n f i b r i l s . F i g u r e A c o n t a i n s a g a l l e y o f e l e c t r o n m i c r o g r a p h s t h a t show t h e s e q u e n t i a l s t a g e s o f t r a n s i t i o n a l o n g t h e d e v e l o p m e n t a l g r a d i e n t a t t h e D E J . A t a d e v e l o p m e n t a l s t a g e between t h o s e shown i n F i g s . 1b and c , remnan ts o f t he BM a r e s t i l l p r e s e n t be tween a p i c a l p r o j e c t i o n s o f a m e l o b l a s t s . In t h e p r e - d e n t i n m a t r i x a l o c u s o f m i n e r a l i z a t i o n a p p e a r s t o have been i n i t i a t e d f rom a s i n g l e p o i n t , p r o b a b l y a m a t r i x v e s i c l e ( F i g . A a ) . The e l e c t r o n d e n s i t y o f c o l l a g e n f i b r i l s a s s o c i a t e d w i t h t h i s l o c u s d e m o n s t r a t e t h e r a d i a l p a t t e r n o f m i n e r a l i z a t i o n . As m i n e r a l i z a t i o n p r o g r e s s e s t h i s l o c u s o f m i n e r a l i z a t i o n w i l l become l a r g e r and c o a l e s c e w i t h o t h e r s l i k e i t t o f o rm a c o n t i n u o u s l a y e r o f m a n t l e d e n t i n seen i n F i g . A b . J u s t b e f o r e t h e a m e l o b l a s t e n t e r s t h e s e c r e t o r y phase t h e s u r f a c e o f d e n t i n a p p e a r s uneven due t o c o l l a g e n f i b r i l s j u t t i n g ou t f rom the s u r f a c e o f m i n e r a l i z e d d e n t i n ( F i g . A b ) . when t h e a m e l o b l a s t b e g i n s t o r e l e a s e enamel m a t r i x on to t he d e n t i n s u r f a c e , t h e m a t r i x a p p e a r s t o b a t h e n e w l y m i n e r a l i z e d c o l l a g e n f i b r i l s (Ularshawsky 1 9 8 5 ) . Enamel c r y s t a l s f o rm on t h e s u r f a c e o f t he d e n t i n and grow i n t o t he s t i p p l e d enamel m a t r i x t o w a r d s t h e a m e l o b l a s t ( F i g s . A c ) . A l o n g t h e DEJ t he d i s t a n c e be tween t h e a m e l o b l a s t l a y e r and t h e s u r f a c e o f t he d e n t i n i s i n c r e a s e d due t o t he s u b s e q u e n t m a t r i x r e l e a s e and c r y s t a l g row th 44 FIGURE 3 - CHANGES IN COLLAGEN A5 THE DEJ FORMS ( a , b ) H igh m a g n i f i c a t i o n e l e c t r o n m i c r o g r a p h s o f t h e a p i c a l r e g i o n o f an a m e l o b l a s t (A) show j a v e l i n p o i n t e d ends o f c o l l a g e n f i b r i l s t o be i n c l o s e p r o x i m i t y t o t he a m e l o b l a s t . (a ) P i n o c y t o t i c - l i k e v e s i c l e s ( l a r g e w h i t e a r rowhead) o b s e r v e d s u g g e s t some m a t r i x r e s o r p t i o n i s t a k i n g p l a c e , (b) T h i s a l t e r a t i o n o c c u r s as m i n e r a l i z a t i o n p r o g r e s s e s f rom t h e d e n t i n (D) t o w a r d s t h e a m e l o b l a s t a l o n g t h e f i b r i l s . ( c ) P r o l o n g e d d e m i n e r a l i z a t i o n and s t a i n i n g e x p o s e s s i m i l a r c o l l a g e n f i b r i l s ( a s t e r i s k s ) t h a t a r e now b o r d e r e d by enamel ( E ) . The ends o f t h e s e f i b r i l s i n d e n t i n (D) have a r e d u c e d a x i a l b a n d i n g , and an expanded f i b r i l l a r a p p e a r a n c e . L u c e n t a r e a s ( s m a l l w h i t e a r r o w h e a d s ) i n t he enamel m a t r i x were f o r m e r l y o c c u p i e d by enamel c r y s t a l s . B a r = 200 nm; Mag = 9 9 , 0 0 0 x . [ c o n v e n t i o n a l l y p r e p a r e d , d e m i n e r a l i z e d w i t h EDTA and s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d ( P T A J / U A ]  46 ( F i g s . 4 d - f ) (Osbo rn 1 9 7 2 ) . A f t e r a u n i f o r m l y t h i c k l a y e r has been fo rmed t h e a m e l o b l a s t d e v e l o p e s t h e Tomes' p r o c e s s , an a p i c a l p r o j e c t i o n w h i c h d i r e c t s t h e o r i e n t a t i o n o f enamel c r y s t a l s i n t o r o d and i n t e r r e d ( F i g s . 4 e - f ) . The -p resence o f a l a r g e number o f e l e c t r o n - d e n s e v e s i c l e s w i t h i n a Tomes' p r o c e s s i n d i c a t e t he l a r g e amount o f enamel m a t r i x w h i c h i s s e c r e t e d d u r i n g t h e f o r m a t i o n o f e n a m e l . S e c t i o n s o f t h e DEJ were d e m i n e r a l i z e d and t h e n s t a i n e d so t h a t a l l d e t e c t a b l e m i n e r a l wou ld be removed so t h a t t h e u n d e r l i n i n g o r g a n i c s t r u c t u r e c o u l d be o b s e r v e d i n b o t h c r y o g e n i c ( F i g . 5a) and c o n v e n t i o n a l ( F i g . 5b) p r e p a r a t i o n s . In b o t h p r e p a r a t i o n s a 5 nm w ide e l e c t r o n l u c e n t s p a c e can be o b s e r v e d where t he enamel c r y s t a l e x i s t e d p r i o r t o d e r n i n e r a l i z a t i o n . The e l e c t r o n - d e n s e m a t e r i a l s u r r o u n d i n g t h e s p a c e i s t h e enamel s h e a t h ( F i g . 5 a ) , o t h e r a u t h o r s have o b s e r v e d t h i s s t r u c t u r e and i t s r e l a t i o n s h i p w i t h enamel c r y s t a l s ( J e s s e n , 1 9 6 8 ; T r a v i s and G l i m c h e r , 1 9 6 4 ) . The enamel s h e a t h a p p e a r s t o be i n t i m a t e l y a s s o c i a t e d w i t h t h e s u r f a c e o f d e n t i n ( F i g . 5 a ) and s p e c i f i c a l l y w i t h t h e f r a y e d end o f a c o l l a g e n f i b r i l ( F i g . 5 b ) . I n d i v i d u a l s h e a t h s e x t e n d f r om t h e c o l l a g e n f i b r i l t o t h e edge o f t h e enamel m a t r i x j u s t be low t h e a m e l o b l a s t c e l l membrane. F i g u r e 6c i s an u n s t a i n e d b r i g h t f i e l d image o f t h e D E J , i t i s a p p r o x i m a t e l y t h e same s t a g e o f deve lopmen t as F i g s . 1 e , 4 f , 5 a , and b . Enamel and d e n t i n a p a t i t e c r y s t a l s a r e imaged h e r e due t o t h e i r e l e c t r o n d e n s i t y , t h e much l e s s e l e c t r o n - d e n s e o r g a n i c m a t r i x i s n o t s e e n . C o n f i r m a t i o n t h a t t h e s e two g r o u p s o f c r y s t a l s a r e a p a t i t e comes f rom t h e i r e l e c t r o n d i f f r a c t i o n p a t t e r n s w h i c h have t he t y p i c a l a p a t i t e d - s p a c i n g s ( F i g s . 6a and b ) . The d i f f r a c t i o n p a t t e r n s o f b o t h 47 FIGURE 4 - SEQUENTIAL STEPS OF ENAMEL MINERALIZATION (a) B e f o r e t he enamel m a t r i x i s r e l e a s e d a l o c u s o f m i n e r a l i z a t i o n can be o b s e r v e d i n t he p r e d e n t i n m a t r i x ( l a r g e a r r o w ) . M i n e r a l i z a t i o n a p p e a r s t o s p r e a d e p i t a x i a l l y a l o n g c o l l a g e n f i b r i l s . P r o c e s s e s o f a m e l o b l a s t s (A) e x t e n d i n t o t h e p r e d e n t i n m a t r i x . (b) As m i n e r a l i z a t i o n r e a c h e s t h e ends o f c o l l a g e n f i b r i l s a t t h e s u r f a c e o f d e n t i n t h e enamel m a t r i x i s r e l e a s e d ( s m a l l a r r o w ) . ( c ) The e l e c t r o n - d e n s e enamel m a t r i x c o v e r s t h e s u r f a c e o f t h e d e n t i n , and soon a f t e r i t i s r e l e a s e d . Enamel c r y s t a l s , w h i c h a p p e a r a s t h i n e l e c t r o n - d e n s e l i n e s , a r e seen t o have e x t e n d e d f rom t h e d e n t i n t owa rd a m e l o b l a s t s . The c r y s t a l s a p p e a r o n l y t o grow i n t o r e g i o n s where t h e r e i s enamel m a t r i x . ( d - f ) F u r t h e r a l o n g t he DEJ more enamel m a t r i x i s s e c r e t e d and t h e a m e l o b l a s t s a p p e a r t o move away f rom t h e s u r f a c e o f d e n t i n . The enamel c r y s t a l s grow t o be i n c l o s e p r o x i m i t y t o the s u r f a c e o f t h e a m e l o b l a s t s ( c u r v e d a r r o w s ) . B a r = 500 nm; Mag = 3 5 , 0 0 0 x . [ c o n v e n t i o n a l l y p r e p a r e d and s t a i n e d w i t h U A / L C ] 48 49 FIGURE 5 - THE ENAMEL SHEATH (a ) A c r y o g e n i c a l l y p r e p a r e d , d e m i n e r a l i z e d / s t a i n e d s e c t i o n o f t h e DEJ shows enamel s h e a t h s w i t h i n enamel ( E ) . These s h e a t h s a r e a s s o c i a t e d w i t h t h e s u r f a c e o f d e n t i n (D) ( l a r g e w h i t e a r r o w h e a d ) . The d i a m e t e r o f t h e s h e a t h i s 5nm a t t h i s s t a g e o f deve lopmen t ( s m a l l b l a c k a r r o w h e a d s ) . (b) A s i m i l a r s e c t i o n c o n v e n t i o n a l l y p r e p a r e d shows t h a t t h e s h e a t h i n enamel (E) i s a s s o c i a t e d w i t h t h e f r a y e d end o f a c o l l a g e n f i b r i l ( l a r g e w h i t e a r rowhead) w i t h i n d e n t i n ( D ) . The d i a m e t e r o f t h e s e s h e a t h s i s 5nm. There a p p e a r s t o be some s p a c e between t h e p l a s m a membrane o f t he a m e l o b l a s t and t h e end o f t he s h e a t h . B a r = 100 nm; Mag = 1 3 2 , 0 0 0 x . [ bo th d e m i n e r a l i z e d w i t h EDTA and s t a i n e d w i t h PTA/UA] 50 51 enamel ( F i g . 6a) and dentin ( F i g . 6b) have maxima of the same spacing i n d i c a t i n g that t h e i r u n i t c e l l dimensions are the same, however the p a t t e r n f o r enamel i s l e s s intense than that of the dentin. This d i f f e r e n c e i s due to the sparse d i s t r i b u t i o n of enamel c r y s t a l s which are 5 nm i n width and vary i n length at t h i s stage as compared to the smaller more densly packed dentin c r y s t a l s . An intimate s p a t i a l r e l a t i o n s h i p between enamel and dentin c r y s t a l s can be observed i n F i g . 6 c . This r e l a t i o n s h i p i s not l i m i t e d to the surface of d e n t i n , but can a l s o be observed i n the d e n t i n a l furrows between co l l a g e n f i b r i l s . M i n e r a l i z e d collagen f i b r i l s appear blunt-ended as they j u t out from the surface of the dentin. A s e l e c t e d - a r e a dark f i e l d (SADF) image of the same area ( F i g . 6d) demonstrates t h i s r e l a t i o n s h i p as w e l l . Enamel c r y s t a l s can be d i s t i n g u i s h e d from dentin c r y s t a l s at the DEJ due to t h e i r l a r g e r s i z e and p o s i t i o n i n g . C r y s t a l c o n t i n u i t y between dentin and enamel i s more c l e a r l y demonstrated with SADF imaging because the image shows only the a p a t i t e . Figure 6d demonstrates at the d i f f e r e n t l o c a t i o n s along the DEJ l a r g e r enamel c r y s t a l s are i n t i m a t e l y associated with the smaller dentin c r y t a l s . Selected-area dark f i e l d can a l s o be used to demonstrate a c r y s t a l o r g a n i z a t i o n i n dentin that i s not apparent i n b r i g h t f i e l d imaging due to i t s heavy m i n e r a l i z a t i o n . Figures 7 a-d give an overview of the mineral component of dentin with d i f f e r e n t modes of imaging, at a developmentally more mature DEJ than i n F i g . 6 . Figure 7a i s an unstained b r i g h t f i e l d image of the DEJ, c r y s t a l s w i t h i n the dentin appear to be randomly arranged, except along h e a v i l y m i n e r a l i z e d c o l l a g e n f i b r i l s . The SADF image of t h i s area formed by the ab+c-axial r e f l e c t i o n s does not add any information to the b r i g h t 52 FIGURE 6 - CRYSTAL CONTINUITY- NEWLY FORMED DENTINO-ENAMEL JUNCTION ( a - b ) D i f f r a c t i o n p a t t e r n s o f newly fo rmed a p a t i t e i n (a ) enamel and (b) d e n t i n demons t ra te t h e maxima ( r e f l e c t e d e l e c t r o n s ) o f t h e c a x i s (1) and a combined ab+c a x e s ( 2 ) . The maxima o f i n i t i a l enamel a p p e a r more f a i n t than t h o s e o f d e n t i n a t t h i s s t a g e o f d e v e l o p m e n t . ( c - d ) Companion b r i g h t f i e l d ( c ) and s e l e c t e d - a r e a da rk f i e l d (d ) images o f t h e DEJ a t a s t a g e s i m i l a r t o F i g u r e s 1e and 4 f . ( c ) O n l y t he h i g h mass d e n s i t i e s o f a p a t i t e c r y s t a l s i n b o t h enamel and d e n t i n a r e v i s u a l i z e d . A m i n e r a l i z e d c o l l a g e n f i b r i l (C) p r o j e c t s i n t o t h e enamel m a t r i x , (d) A combined a x i a l image shows a c l o s e a s s o c i a t i o n between d e n t i n and enamel a p a t i t e c r y s t a l s . A r rows ( b l a c k and w h i t e ) d e l i n e a t e t h i s c l o s e s p a t i a l r e l a t i o n s h i p between d e n t i n and enamel c r y s t a l s . Ba r = 100 nm; Mag = 1 3 8 , 0 0 0 x . [ c r y o g e n i c a l l y p r e p a r e d ; u n s t a i n e d ] , ( images c and d c o u r t e s y o f D r . A . L . A r s e n a u l t )  54 FIGURE 7 - CRYSTAL ORGANIZATION IN DENTIN (a ) A b r i g h t f i e l d image o f t h e DEJ a t a l a t e r s t a g e o f deve lopmen t d e m o n s t r a t e s m i n e r a l i z e d c o l l a g e n f i b r i l s (C) w i t h i n d e n t i n (D) and l a r g e r a p a t i t e c r y s t a l s w i t h i n t h e enamel ( E ) . (b) T h i s same a r e a i s shown i n an a , b + c - a x i a l da rk f i e l d image w h i c h d e m o n s t r a t e s t h e d i s t r i b u t i o n o f a p a t i t e c r y t a l s w i t h i n d e n t i n and e n a m e l . ( c - d ) These da rk f i e l d images s e l e c t i v e l y d i s c r i m i n a t e a , b - a x e s and c - a x i s . The numbers ( 1 - 3 ) c o r r e s p o n d t o t h e d i f f e r e n t o r i e n t a t i o n s o f c r y s t a l s w i t h i n d e n t i n . The c r y s t a l o r g a n i z a t i o n i n t h e c - a x i s da rk f i e l d image (d) c o r r e s p o n d s t o t h e m i n e r a l i z e d c o l l a g e n f i b r i l s i n t h e b r i g h t f i e l d image ( a ) . B a r = 200 nm; Mag = 4 7 , 5 0 0 x . [ c r y o g e n i c a l l y p r e p a r e d ; u n s t a i n e d ] 55 56 f i e l d image ( F i g . 7 b ) . The da rk f i e l d a b - a x e s image shows a m o s t l y random d i s t r i b u t i o n o f c r y s t a l s and some s p a c e s where t h e r e a r e no c r y s t a l s , t h e s e c o r r e s p o n d t o a r e a s o f c - a x i a l l y o r i e n t e d c r y s t a l s i n F i g . 7 d . F i g u r e 7d i s a da rk f i e l d image made up o f r e f l e c t e d e l e c t r o n s f r om the c - a x i s l a t t i c e p l a n e o f a p a t i t e c r y s t a l s w h i c h a r e l y i n g p e r p e n d i c u l a r t o t r a n s m i t t e d beam. D e n t i n a p a t i t e c r y s t a l s a r e s e e n t o be o r i e n t e d a l o n g some c o l l a g e n f i b r i l s , bu t no t o t h e r s . C u r v i n g o f t h e f i b r i l s ou t o f t h e p l a n e o f t h e image , o v e r l a y i n g o f o t h e r f i b r i l s o r changes o f o r i e n t a t i o n o f c r y s t a l s w i t h i n t h e c o l l a g e n c o u l d have t h e s e r e s u l t s . I t s h o u l d be n o t e d t h a t SADF images o n l y a p o r t i o n o f t h e t o t a l a p a t i t e c r y s t a l d i s t r i b u t i o n w i t h i n d e n t i n and enamel because t h e o b j e c t i v e a p e r t u r e wh ich i s used t o s e l e c t a p a t i t e l a t t i c e r e f l e c t i o n s f r om t h e d i f f r a c t i o n p a t t e r n i s o f l i m i t e d d i a m e t e r , t h e r e f o r e i t s e l e c t s o n l y a s m a l l p o r t i o n o f t h e maxima o f r e f l e c t i o n s t h a t r e s u l t f rom a random o r g a n i z a t i o n o f c r y s t a l s . M a t u r a t i o n i s o b s e r v e d i n l a t e r d e v e l o p m e n t a l s t a g e s by t h e i n c r e a s e d w i d t h o f enamel c r y s t a l s ( F i g . 8 a ) . M a t u r a t i o n i s a p r o c e s s whereby t he amount o r g a n i c m a t r i x i s r e d u c e d w h i l e t h e i n o r g a n i c m a t e r i a l i s i n c r e a s e d . These w i d e r enamel c r y s t a l s (25 nm w i d e ) c a n e a s i l y be d i s t i n g u i s h e d f rom the s m a l l e r d e n t i n c r y s t a l s (5 nm w i d e ) . As i n t h e l e s s mature enamel ( F i g . 6 a ) , t h e s e enamel c r y s t a l s a r e i n an i n t i m a t e s p a t i a l r e l a t i o n s h i p w i t h d e n t i n c r y s t a l s , n o t o n l y i n t he h e a v i l y m i n e r a l i z e d c o l l a g e n f i b r i l s , b u t a l s o i n between t h e s e f i b r i l s w i t h i n d e n t i n f u r r o w s . SADF, u s i n g r e f l e c t i o n s f rom t h e a b + c - a x e s , o f t h e DEJ a t t h e same s t a g e a l s o d e m o n s t r a t e s p a t i a l i n t i m a c y between enamel and d e n t i n c r y s t a l s ( F i g . 8 b - d ) . 57 FIGURE 8 - CRYSTAL CONTINUITY- MORE MATURE DENTINO-ENAMEL JUNCTION (a ) As t h e DEJ ma tu res t h e c r y s t a l s w i t h i n enamel (E) i n c r e a s e i n s i z e . They a r e o b s e r v e d i n t h i s b r i g h t f e i l d image t o be a s s o c i a t e d w i t h t he d e n t i n (D) n o t o n l y i t s s u r f a c e ( l a r g e a r r o w ) , b u t a l s o deep w i t h i n d e n t i n a l f u r r o w s between m i n e r a l i z e d c o l l a g e n f i b r i l s (C) ( s m a l l a r r o w s ) . ( b - d ) A combined a x i a l image d e m o n s t r a t e s t he same c r y s t a l c o n t i n u i t y between a p a t i t e c r y s t a l s o f d e n t i n and enamel as o b s e r v e d a t t h e newly fo rmed DEJ ( F i g . 6 d ) . The c l o s e s p a t i a l r e l a t i o n s h i p be tween d e n t i n and enamel c r y s t a l s ( w h i t e a r r o w h e a d s ) i s c l e a r l y e v i d e n t b e c a u s e o f t h e g r e a t e r d i f f e r e n c e i n c r y s t a l s i z e a t t h i s s t a g e o f d e v e l o p m e n t . B a r - 100 nm; Mag = 9 3 , 0 0 0 . [ a i r d r i e d ; u n s t a i n e d ] 58 59 FIGURE 9 - SCHEMATIC REPRESENTATION DF DENTINO-ENAMEL JUNCTION FORMATION T h i s i s a s c h e m a t i c r e p r e s e n t a t i o n o f t h e ma jo r e v e n t s l e a d i n g t o and i n c l u d i n g t h e i n i t i a t i o n o f enamel m i n e r a l i z a t i o n , (a ) P r i o r t o m i n e r a l i z a t i o n o f d e n t i n a m e l o b l a s t s (A) w h i c h c o n t a i n p r o t e i n s e c r e t o r y a p p a r a t u s a r e a s s o c i a t e d w i t h a b a s a l l a m i n a ( B L ) . W i t h i n t h e p r e - d e n t i n (PD) c o l l a g e n f i b r i l s (C) o c c u r i n a random a r r a n g e m e n t , between them a r e m a t r i x v e s i c l e s (MU) and o d o n t o b l a s t p r o c e s s e s ( O P ) . (b) M i n e r a l i z a t i o n o f d e n t i n (D) i s i n i t i a t e d a f t e r t h e breakdown and p a r t i a l r e m o v a l o f t h e b a s a l l a m i n a . I n i t i a t i o n o f d e n t i n m i n e r a l i z a t i o n a r i s e s w i t h i n m a t r i x v e s i c l e s and t h e e p i t a x i a l s p r e a d o f d e n t i n a p a t i t e c r y s t a l s f o l l o w s c o l l a g e n f i b r i l s a s i t p r o c e e d s t owa rd a m e l o b l a s t s . Once m i n e r a l i z a t i o n r e a c h e s t he edge o f d e n t i n , a m e l o b l a s t s b e g i n t o e x o c y t o s e t h e enamel m a t r i x w h i c h has been s t o r e d i n s e c r e t o r y v e s i c l e s (S\7) . P i n o c y t o t i c v e s i c l e s (P\l) t h a t , a p p e a r a t t h e a p i c a l membrane o f a m e l o b l a s t s a r e t h o u g h t t o be i n v o l v e d i n t h e breakdown and r e s o r p t i o n o f t h e b a s a l l a m i n a . They may a l s o have a r o l e i n r e s o r b i n g t h e t e r m i n a l p o r t i o n s o f p r e - d e n t i n c o l l a g e n f i b r i l s a s s o c i a t e d w i t h t he b a s a l l a m i n a . ( c ) Enamel a p a t i t e c r y s t a l s (EC) grow f rom the edge o f d e n t i n t h r o u g h t h e enamel m a t r i x ( E M ) , v t owa rd a m e l o b l a s t s . (d) The a r e a bounded by a box i n ( c ) i s m a g n i f i e d t o show the i n t e r p r e t e d r e l a t i o n s h i p between d e n t i n and e n a m e l . T h i s p l a t e i s a w o r k i n g model o f t he s p a t i a l r e l a t i o n s h i p be tween b o t h t he o r g a n i c and t he i n o r g a n i c components o f d e n t i n and e n a m e l . In t h i s f i g u r e t h e c o l l a g e n m o l e c u l e s o f t he f r a y e d c o l l a g e n 60 f i b r i l a p p e a r s t o be p r e s e n t i n g d e n t i n a p a t i t e c r y s t a l s (DC) t o t h e enamel m a t r i x . Some o f t h e s e d e n t i n c r y s t a l s become enamel c r y s t a l s as t h e y f o rm w i t h i n enamel s h e a t h ( r e d ) . The i n t i m a t e s p a t i a l r e l a t i o n s h i p between d e n t i n and enamel c r y s t a l s i s d e l i n e a t e d by a l a r g e a r r o w h e a d . 61 62 DISCUSSION The purpose of t h i s t h e s i s which examines the formation of the dentino-enamel j u n c t i o n (DEJ) was to f i n d out how b i o l o g i c a l a p a t i t e c r y s t a l s of enamel are i n i t i a l l y formed. Information from other authors suggests that the i n i t i a t i o n of enamel formation i s d i f f e r e n t from both dentin and bone because the enamel matrix does not contain matrix v e s i c l e or collagen f i b r i l s , as w e l l dentin i s shown to form p r i o r to enamel, but the i n i t i a t i n g mechanism has not been proposed (Eisenmann and G l i c k , 1972; R e i t h , 1967; Ronnholm, 1962). Of a l l the authors who have presented an e l e c t r o n microscopic (EM) view of the development of the DEJ, only Bernard (1972) has suggested that enamel m i n e r a l i z a t i o n occurs as a secondary m i n e r a l i z a t i o n process which i s r e l i a n t upon dentin m i n e r a l i z a t i o n based upon evidence of a c l o s e s p a t i a l and temporal r e l a t i o n s h i p between c r y s t a l s of dentin and .enamel. E x t r a p o l a t i o n of h i s hypothesis leads to the suggestion that enamel c r y s t a l s may be extensions of dentin c r y s t a l s which grow i n t o the enamel matrix. Evidence f o r t h i s extended hypothesis which would be a d i r e c t c r y s t a l c o n t i n u i t y between dentin and enamel c r y s t a l s i s examined i n t h i s t h e s i s . In t h i s t h e s i s I w i l l show evidence that t h i s c o n t i n u i t y e x i s t s and present a model to summarize the involvement of both dentin and enamel organic matrices i n p r o v i d i n g an environment conducive to forming c r y s t a l c o n t i n u i t y ( F i g . 9). Before d e s c r i b i n g how the DEJ i s formed the importance of the i n t i m a t e s p a t i a l r e l a t i o n s h i p between dentin and enamel must be e s t a b l i s h e d by presenting some f u n c t i o n a l p o s s i b i l i t i e s from t h i s i n t e r a c t i o n . Dentin may provide a s t r u c t u r a l foundation onto which 63 t h e enamel can f o r m , a c h e m i c a l b a n d i n g may o c c u r between t h e two o r g a n i c m a t r i c e s w h i l e c r y s t a l c o n t i n u i t y i s e s t a b l i s h e d . The f i r m a t t a c h m e n t o f enamel t o d e n t i n i s r e q u i r e d f rom the t o o t h ' s f u n c t i o n i n m a s t i c a t i o n . D e n t i n may be t o t he enamel as m a t r i x v e s i c l e s a r e t o d e n t i n , a c t i n g as a n u c l e a t i n g a g e n t f o r enamel c r y s t a l s ; o r g a n i c / i n o r g a n i c components o f d e n t i n may a l t e r t he i n i t i a l l y r e l e a s e d enamel m a t r i x so t h a t i t w i l l m i n e r a l i z e . A l t e r a t i o n o f t h e p r e d e n t i n m a t r i x a t t he d e n t i n o - a m e l o b l a s t j u n c t i o n (DA j ) p r i o r t o enamel m a t r i x r e l e a s e may a l l o w the d i r e c t e x p o s u r e o f d e n t i n c r y s t a l s t o t he enamel m a t r i x p r o m o t i n g an e p i t a x i a l g rowth o f enamel c r y s t a l s . Enamel c r y s t a l s w h i c h i n t e r d i g i t a t e w i t h t h e uneven s u r f a c e o f d e n t i n wou ld a l s o promote a s t r o n g a t t a c h e m e n t o f enamel t o d e n t i n . These f u n c t i o n a l p o s s i b i l i t i e s show a r e l i a n c e o f enamel on t h e n o r m a l f o r m a t o n o f d e n t i n , t h i s i s e v i d e n t i n p a t h o l o g i c a l c o n d i t i o n s where d e n t i n a l d y s p h a s i a s ( d e n t i n o g e n e s i s i m p e r f e c t a , o p a l e s c e n t d e n t i n , and s h e l l t e e t h ) a f f e c t enamel f o r m a t i o n o r i t s a t t a c h e m e n t t o d e n t i n ( R u s h t o n , 1 9 5 4 ; S u z u k i e t a l . , 1 9 7 7 ; L e v i n e t a l . , 1 9 8 3 ; C l e r g e a u - G u e r i t h a u l t and J a s m i n , 1 9 8 5 ) . A c a s e s t u d y o f a d e n t a l d y s p h a s i a , o p a l e s c e n t d e n t i n , showed t h e DEJ t o be a s t r a i g h t l i n e r a t h e r t h a n i t s r e g u l a r s c a l l o p e d a p p e a r a n c e , t h e l a y e r o f enamel was h y p o m i n e r a l i z e d and much t h i n n e r t h a n i n t h e n o r m a l c o n d i t i o n i t a l s o l a c k e d t he r o d / i n t e r r o d a r rangemen t o f c r y s t a l s ( S u s u k i e t a l . , 1 9 7 7 ) . These a b n o r m a l i t i e s o f t he enamel were i n a d d i t i o n t o t h e p o o r l y fo rmed d e n t i n o f t h i s c o n d i t i o n . I n many o f t h e s e d y s p h a s i a s t h e r e a r e l e s s s e v e r e v a r i a n t s where t h e enamel l a y e r a p p e a r s n o r m a l , t h i s o n l y o c c u r s i n c o n j u n c t i o n w i t h t h e n o r m a l f o r m a t i o n o f t h e m a n t l e l a y e r o f d e n t i n ( W i t k o p , 1 9 7 1 ) . 64 The developmental sequences of dentin and enamel are presented i n t h i s t h e s i s i n the f o l l o w i n g order of events. (1 ) Matrix v e s i c l e s w i t h i n pre-dentin that i n i t i a t e m i n e r a l i z a t i o n contain c r y s t a l - l i k e electron-dense m a t e r i a l . These matrix v e s i c l e s are p o s i t i o n e d between c o l l a g e n f i b r i l s that are randomly arranged; the basal lamina between pre-dentin and ameloblasts i s i n t a c t ( F i g s . 1b and 2b). (2) The basal lamina i s penetrated by ameloblast c e l l processes and there i s an increase i n the amount of a p a t i t e c r y s t a l s i n the area where the i n i t i a l matrix v e s i c l e s were. M i n e r a l i z i n g regions i n pre-dentin are described as l o c i (Hayashi, 1983; 1984; F i g s . 1c and 4a). (3) As more c r y s t a l s form, there i s a spread of m i n e r a l i z a t i o n from i n i t i a l l o c i i n a f i b r i l l a r regions to collagen f i b r i l s , and then e p i t a x i a l l y along these making the l o c i l a r g e r ( F i g s . 2d, e and 4a). (4) M i n e r a l i z a t i o n proceeds along collagen f i b r i l s toward ameloblasts which begin to secrete an e l e c t r o n dense product which has been synthesized and st o r e d i n v e s i c l e s near the a p i c a l c e l l s urface. The enamel matrix covers the surface of dentin ( F i g . 4b). (5) Soon a f t e r the enamel matrix i s released a p a t i t e c r y s t a l s form on the surface of the dentin and begin to grow toward the ameloblast, only i n areas where the enamel matrix i s present ( F i g s . 1d and 4 c ) . (6) As more matrix i s released the enamel c r y s t a l s grow to be i n c l o s e proximity to the a p i c a l surface of the ameloblast ( F i g s . 4d-f) ( R e i t h , 1967; Bernard, 1972;^ Eisenman and G l i c k , 1972; Katchburian, 1973, S l a v k i n and Bringas, 1976). These events suggest that there i s a progression of m i n e r a l i z a t i o n i n i t i a t e d i n dentin and continued i n enamel. Collagen may have a r o l e i n presenting mineral i n dentin to enamel because 65 m i n e r a l a s s o c i a t e d w i t h c o l l a g e n f i b r i l s i s t h e f i r s t t o come i n c o n t a c t w i t h new r e l e a s e d enamel m a t r i x ( F i g s . 1 c , 2 e , and 4 a - c ) , and t h e f i b r i l s a r e known t o o r g a n i z e a p a t i t e c r y s t a l s w i t h t h e i r c - a x i s p a r a l l e l t o t h e c - a x i s o f t h e f i b r i l ( F i g . 7 ; A r s e n a u l t , 1 9 8 8 ) . The a c t i v e i n v o l v e m e n t o f d e n t i n c o l l a g e n f i b r i l s i n t h e p r e s e n t a t i o n o f d e n t i n a p a t i t e t o t h e enamel m a t r i x may be r e l a t e d t o c h a n g e s i n t h e a p p e a r a n c e o f t e r m i n a l p o r t i o n s o f c o l l a g e n f i b r i l s d u r i n g t he f o r m a t i o n o f t h e D E J . B e f o r e c o m p l e t e m i n e r a l i z a t i o n o f p r e d e n t i n t h e t e r m i n a l p o r t i o n s o f c o l l a g e n f i b r i l s a s s o c i a t e d w i t h t h e r e t i c u l a r l a m i n a o f t h e BM have been d e s c r i b e d a s j a v e l i n - l i k e i n a p p e a r a n c e ( F i g s . 1 b , 2 a - c , and ha; B e r n a r d , 1 9 7 2 ) . A f t e r t h e BM i s removed t h e s e f i b r i l s a r e c l o s e l y a s s o c i a t e d w i t h t h e a p i c a l s u r f a c e o f a m e l o b l a s t s w h i c h e x t e n d i n t o t h e p r e d e n t i n m a t r i x ( F i g . 3 a , and b ) . A f t e r m i n e r a l i z a t i o n t h e t e r m i n a l p o r t i o n s o f d e n t i n c o l l a g e n f i b r i l s d r a m a t i c a l l y c h a n g e ; r e m o v a l o f m i n e r a l f rom a l o n g i t u d i n a l t h i n s e c t i o n o f t h e DEJ show t h e s e f i b r i l s t o be f r a y e d a t t h e ends p r e v i o u s l y a s s o c i a t e d w i t h the BM. I t a p p e a r s a s i f t h e i n t e r m o l e c u l a r c r o s s - l i n k s between c o l l a g e n m o l e c u l e s b reak t o g i v e t h e u n r a v e l l e d a p p e a r a n c e o f t he t e r m i n a l p o r t i o n s o f t h e s e c o l l a g e n f i b r i l s i n t h e p r e s e n c e o f t h e enamel m a t r i x . These o b s e r v e d changes t o t h e s e c o l l a g e n f i b r i l s may r e s u l t f rom a change i n t h e i r f u n c t i o n a l s i g n i f i c a n c e . P r i o r t o m i n e r a l i z a t i o n , j a v e l i n - p o i n t e d c o l l a g e n f i b r i l s may have a r o l e i n a n c h o r i n g t h e BM g i v i n g t h e o d o n t o b l a s t s , w h i c h p r o d u c e t h i s c o l l a g e n , an i n d i r e c t c o n t r o l o v e r t h e o r i e n t a t i o n o f a m e l o b l a s t s w h i c h r e s t on the BM ( B e r n a r d , 1 9 7 2 ) . T h i s a n c h o r i n g o f c o n n e c t i v e t i s s u e s t o basement membranes p r o d u c e d by e p i t h e l i a l c e l l s has been documented i n o t h e r t i s s u e s y s t e m s i n a r e c e n t s t u d y by 66 Inoue and L e b l o n d ( 1 9 8 8 ) . A f t e r the BM has been removed t he a n c h o r i n g o f a m e l o b l a s t s by c o l l a g e n f i b r i l s a p p e a r s t o c o n t i n u e u n t i l t h e ename l m a t r i x i s s e c r e t e d ( F i g s . 3a and b ) , bu t d u r i n g t h i s m i n e r a l i z a t i o n o f t h e d e n t i n m a t r i x t e r m i n a l p o r t i o n s o f c o l l a g e n f i b r i l s become f r a y e d i n a p p e a r a n c e ( F i g s . 3c and 5 b ) . The r o l e o f t h e s e r e m o d e l l e d c o l l a g e n f i b r i l s may be i n p r e s e n t i n g a p a t i t e o f d e n t i n t o t h e enamel m a t r i x w h i c h i s r e l e a s e d upon them, b u t t he f r a y e d c o l l a g e n f i b r i l s may a l s o c o n t r i b u t e t o t h e f o r m a t i o n o f a s t r o n g bond between t he d e n t i n and enamel a t t h e D E J . A p o s s i b l e e x p l a n a t i o n f o r t h e changes o c c u r r i n g t o t h e t e r m i n a l p o r t i o n s o f t h e s e c o l l a g e n f i b r i l s i s t h a t o d o n t o b l a s t d e r i v e d c o l l a g e n a s e , s t o r e d i n m a t r i x v e s i c l e s i n p r e - d e n t i n , i s r e l e a s e d d u r i n g m i n e r a l i z a t i o n o f d e n t i n and d e g r a d e s - t h e ends o f t he c o l l a g e n f i b r i l s g i v i n g them t h e i r f r a y e d a p p e a r a n c e . E v i d e n c e f o r t h i s e x p l a n a t i o n i s p r o v i d e d by a s t u d y w h i c h f o u n d c o l l a g e n a s e a c t i v i t y a s s o c i a t e d w i t h t h e c o n t e n t s o f m a t r i x v e s i c l e s i s o l a t e d f rom t h e p r e d e n t i n m a t r i x o f r a b b i t i n c i s o r t o o t h o r g a n s ( S o r g e n t e e t a l , 1 9 7 7 ) . The c o n t e n t s o f t h e s e i s o l a t e d m a t r i x v e s i c l e s have t h e a b i l i t y t o degrade c o l l a g e n m o l e c u l e s i n t o two m o l e c u l a r s p e c i e s b a s e d upon t h e i r m o l e c u l a r w e i g h t s , l i k e o t h e r mammalian c o l l a g e n a s e s . S o r g e n t e e t a l (1977) s u g g e s t e d t h a t t h i s a c t i v i t y i s i n v o l v e d i n t h e breakdown o f t he c o l l a g e n t y p e IV m o l e c u l e s w i t h i n t h e b a s a l l a m i n a , w h i c h may be t he c a s e bu t i n o r d e r t o show t h e enzyme a c t i v i t y o f t h e s e m a t r i x v e s i c l e s , t y p e I c o l l a g e n was used as a s u b s t r a t e . The c o l l a g e n a s e o f d e n t i n m a t r i x v e s i c l e s c o u l d be i n v o l v e d i n d e g r a d i n g t h e t e r m i n a l p o r t i o n s o f t y p e I c o l l a g e n a s w e l l a s t h e b a s a l l a m i n a . R e l e a s e o f c o l l a g e n a s e may be s i m i l a r t o K a t c h b u r i a n ' s (1973) p r o p o s e d 67 mechan ism f o r l e a k a g e and g r a d u a l b reakdown o v e r t i m e o f t h e p l a s m a membrane o f m a t r i x v e s i c l e s i n v o l v e d i n i n i t i a l m i n e r a l i z a t i o n . I t i s more l i k e l y t h a t the enzymes a r e r e l e a s e d when t h e p l a s m a membrane i s p u n c t u r e d by a p a t i t e c r y s t a l s d u r i n g t h e r a p i d m i n e r a l i z a t i o n o f p r e d e n t i n . C o l l a g e n a s e can o n l y a c t on t he n o n - m i n e r a l i z e d c o l l a g e n , and so t he o n l y n o n - m i n e r a l i z e d c o l l a g e n a t t h e enzyme r e l e a s e i s t h e t e r m i n a l p o r t i o n . I f t h e c o l l a g e n a s e was r e l e a s e d p r i o r t o t h i s b u t d i d no t deg rade t h e c o l l a g e n , i t i s p o s s i b l e t h a t t h e enzymes a r e l a t e n t c o l l a g e n a s e s , w h i c h can be a c t i v a t e d by enzymes f r om o t h e r m a t r i x v e s i c l e s ( E e c k o u t and V a e s , 1 9 7 7 ; Sakamoto e t a l , 1 9 8 5 ) . Once c o l l a g e n i s deg raded i t t h e n may be t a k e n up by t h e a m e l o b l a s t , a l o n g w i t h t h e b a s a l l a m i n a ( B L ) . E v i d e n c e f o r t h i s u p t a k e may be t h e a p p e a r a n c e o f many p i n o c y t o t i c v e s i c l e s a l o n g t h e a p i c a l s u r f a c e o f t h e a m e l o b l a s t ( F i g . 3 a ; S l a v k i n and B r i n g a s , 1 9 7 6 ) . W h i l e t h e c o l l a g e n a s e a c t i v i t y i s d e g r a d i n g t h e t e r m i n a l p o r t i o n s o f c o l l a g e n f i b r i l s , m i n e r a l s t i l l a d v a n c e s a l o n g t h e f i b r i l s t owa rd t h e a m e l o b l a s t , wh i ch b e g i n t o r e l e a s e enamel m a t r i x . Warshawsky (1985) b e s t d e s c r i b e d t h i s i n t e r a c t i o n o f t h e enamel and d e n t i n m a t r i c e s by s a y i n g t h a t the enamel m a t r i x " b a t h e s " t h e s u r f a c e o f d e n t i n . The i n t e r a c t i o n between p a r t i a l l y m i n e r a l i z e d c o l l a g e n f i b r i l s o f d e n t i n and t h e n o n - m i n e r a l i z e d enamel m a t r i x may be t h a t t he f r a y e d t e r m i n a l p o r t i o n s ' o f c o l l a g e n f i b r i l s a r e suspended i n t h e enamel m a t r i x p r o v i d i n g an i n t i m a t e a s s o c i a t i o n between t h e s e h e t e r o t y p i c m a t r i c e s where d e n t i n a p a t i t e can be p r e s e n t e d d i r e c t l y t o t h e enamel m a t r i x . S e l e c t e d - a r e a da rk f i e l d i m a g i n g has shown t h a t t h e r e i s an i n t i m a t e a s s o c i a t i o n between d e n t i n and enamel c r y s t a l s i n b o t h i n i t i a l and more mature a r e a s o f t he DEJ ( F i g s . 6d» and 8 b - d ) . T h i s 68 d i r e c t c o n t a c t between d e n t i n and enamel s u g g e s t s e n a m e l ' s r e l i a n c e upon d e n t i n f o r i t s m i n e r a l i z a t i o n , h o w e v e r , f u r t h e r s t u d i e s may be r e q u i r e d t o c o n f i r m t h i s d i r e c t c r y s t a l c o n t i n u i t y . The d e t e r m i n a t i o n o f an i r r e f u t a b l e c r y s t a l r e l a t i o n s h i p between d e n t i n and enamel may p r o v e t o be d i f f i c u l t b e c a u s e o f t h e many t e c h n i c a l l i m i t a t i o n s a s s o c i a t e d w i t h e l e c t r o n m i c r o s c o p y and b i o l o g i c a l a p a t i t e . Some o f t h e s e l i m i t a t i o n s i n c l u d e s p a t i a l o v e r p r o j e c t i o n and t r u n c a t i o n o f t h e s m a l l c r y s t a l s w i t h i n t h i n s e c t i o n s ( H u n z i k e r e t a l . , 1 9 8 9 ) , l i m i t e d d e t e c t i o n o f s m a l l c r y s t a l s o r s p a r s e l y d i s t r i b u t e d c r y s t a l s w h i c h have few l a t t i c e r e p e a t s , and f r a c t u r e o r d i s p l a c e m e n t o f t h e c r y s t a l s w h i c h may o c c u r d u r i n g s e c t i o n i n g . In a d d i t i o n , a p a t i t e c r y s t a l s a r e h i g h l y s u s c e p t i b l e t o r a d i a t i o n damage i n t he e l e c t r o n m i c r o s c o p e . T h i s damage i s most n o t i c e a b l e i n enamel c r y s t a l s w h i c h have a c e n t r a l da rk l i n e a n d / o r a h o l e y a p p e a r a n c e a f t e r o n l y a s h o r t t i m e under t h e e l e c t r o n beam ( K e r e b e l e t a l , 1 9 7 9 ; UJarshawsky and N a n c i , 1 9 8 2 ) . I f i t i s assumed t h a t d e n t i n a p a t i t e c r y s t a l s a r e e x p o s e d t o and grow i n t o t he enamel m a t r i x , t h e n t h e r e must be a d i f f e r e n c e i n t h e mechanisms by wh ich t h e m a t r i x c o n t r o l s c r y s t a l s i z e . The enamel m a t r i x a p p e a r s t o have a d i f f e r e n t c o n t r o l mechanism f o r c r y s t a l g row th f rom d e n t i n a s d e n t i n c r y s t a l s w h i c h grow i n t o t h e enamel m a t r i x a r e no t r e s t r i c t e d t o a u n i f o r m s i z e . A l l o t h e r s k e l e t a l t i s s u e s wh ich c o n t a i n m a t r i x v e s i c l e s and c o l l a g e n ( c a l c i f i e d c a r t i l a g e , b o n e , c a l c i f i e d t u r k e y t e n d o n , and d e n t i n ) have a p a t i t e c r y s t a l s w i t h a p p r o x i m a t e l y t h e same d i m e n s i o n s o f 1 1 - 17 x 5 x 5 nm a s d e t e r m i n e d by SADF, and X - r a y d i f f r a c t i o n ( A r s e n a u l t , 1 9 8 9 ; A r s e n a u l t and G r y n p a s , 1 9 8 8 ; J e n s e n and H o l l e r , 1 9 4 8 ) . I n i t i a l enamel c r y s t a l s , a l t h o u g h somewhat l o n g e r t h a n d e n t i n c r y s t a l s appea r t o have 69 s i m i l a r w i d t h s , and t h i c k n e s s e s ( K e r e b e l e t a l , 1 9 7 9 ) , b u t a s m a t u r a t i o n o c c u r s t he c r y s t a l s grow u n t i l t h e r e i s l i t t l e s p a c e be tween them, t h i s u n l i m i t e d g rowth can be a t t r i b u t e d t o t he u n i q u e enamel m a t r i x ( R o b i n s o n e t a l . , 1 9 8 3 ) . As enamel c r y s t a l s a r e i n t i m a t e l y a s s o c i a t e d w i t h d e n t i n c r y s t a l s , t h e enamel s h e a t h s w h i c h s u r r o u n d enamel c r y s t a l s appea r t o be a s s o c i a t e d w i t h the s u r f a c e s o f d e n t i n c o l l a g e n f i b r i l s ( F i g s . 3 c , 5 a , b and 9 d ) . There may be s e v e r a l ways i n wh ich t he s h e a t h i s i n v o l v e d i n t he f o r m a t i o n o f enamel c r y s t a l s o r t he c o n t i n u e d g rowth o f d e n t i n c r y s t a l s . X - r a y d i f f r a c t i o n s t u d i e s have d e m o n s t r a t e d t h a t t h e e n a m e l i n s , a h e t e r o g e n e o u s g roup o f g l y c o p r o t e i n s t h a t make up t h e enamel s h e a t h , have a b e t a p l e a t e d s h e e t c o n f o r m a t i o n w h i c h a p p r o x i m a t e s t he enamel c r y s t a l s u r f a c e ( G l i m c h e r e t a l . , 1 9 6 1 ; J o d a i k i n e t a l . , 1987 ; 1988 ; T raub e t a l . , 1 9 8 5 ) . These s t u d i e s s u g g e s t t h a t t h e s h e a t h may p r o v i d e some s t r u c t u r a l s u p p o r t f o r t h e c r y s t a l by a l i g n i n g p h o s p h a t e g r o u p s t o be u t i l i z e d i n a p a t i t e f o r m a t i o n i n an o r g a n i z a t i o n s i m i l a r t o t h e u n i t c e l l d i m e n s i o n s o f t h e , c r y s t a l , so t h a t i t e x t e n d s a l o n g i t s c - a x i s . The s h e a t h may a l s o have an a c t i v e r o l e i n t h e f o r m a t i o n o f enamel c r y s t a l s b e c a u s e i t can be r e m i n e r a l i z e d i n t he p r e s e n c e o f c a l c i u m c h l o r i d e , a f t e r d e m i n e r a l i z a t i o n (T raub e t a l . , 1 9 8 5 ) , and e l e c t r o n s p e c t r o s c o p i c a n a l y s i s . has d e m o n s t r a t e d a c o - l o c a l i z a t i o n o f c a l c i u m and p h o s p h a t e t h r o u g h o u t t he t h i c k n e s s o f t h e s h e a t h ( A r s e n a u l t and R o b i n s o n , 1 9 8 9 ) . T h e r e a r e some s i m i l a r i t i e s be tween t h e amino a c i d c o m p o s i t i o n s o f t h e e n a m e l i n s w h i c h make up t he enamel s h e a t h and t he p o s p h o r y n s w h i c h a r e t h o u g h t t o have bo th an i n d u c t i v e and an i n h i b i t o r y r o l e i n d e n t i n m i n e r a l i z a t i o n ; t he ma jo r amino a c i d s o f b o t h a r e s e r i n e and 70 a s p a r t a t e (Te rm ine e t a l . , 1 9 7 9 ; D i m u z i o and V e i s , 1 9 7 8 ) . The e n a m e l i n s a p p e a r no t t o have t he r e s t r i c t i v e i n f l u e n c e on c r y s t a l g r o w t h t h a t t he p h o s p h o r y n s e x h i b i t o v e r d e n t i n c r y s t a l s ( L u s s i e t a l . , 1 9 8 8 ) . The c o n t i n u i t y t h a t e x i s t s between t he o r g a n i c m a t r i c e s o f d e n t i n and enamel may be between c o l l a g e n - a s s o c i a t e d p h o s p h o r y n s and t h e e n a m e l i n s o f t h e enamel s h e a t h . The phenomenon o f a c r y s t a l s h e a t h has a l s o been o b s e r v e d i n i n v e r t e b r a t e s . A d d a d i and UJeiner (1985) show t h a t a c i d i c m a t r i x m a c r o m o l e c u l e s , e x t r a c t e d f rom m o l l u s k s , fo rmed a b e t a p l e a t e d s h e e t r i c h i n a s p a r t i c a c i d wh i ch i n t e r a c t s w i t h t h e c r y s t a l c o n t r o l l i n g t h e p a t t e r n and r a t e o f c a l c i t e c r y s t a l g r o w t h . The p r o t e i n c o m p o s i t i o n o f t h e s e m a c r o m o l e c u l e s i s s i m i l a r t o t h o s e o f e n a m e l i n s . I t a p p e a r s t h a t t h e s h e a t h a c t s t o c o m p a r t m e n t a l i z e a g r o w i n g c r y s t a l by p r o v i d i n g n o t o n l y a l o c a l i z e d e n v i r o n m e n t f o r c o n t i n u o u s c r y s t a l g r o w t h , bu t a l s o i n p r o t e c t i n g t h e a m e l o b l a s t f rom d i r e c t c r y s t a l c o n t a c t . ( F i g s . 4 d - f ) . T h i s l a s t f u n c t i o n o f t he s h e a t h does no t a p p e a r t o be needed i n o t h e r m i n e r a l i z e d t i s s u e s b e c a u s e t h e r e i s a l w a y s a l a y e r o f e x t r a c e l l u l a r m a t r i x between t he c e l l s and t h e m i n e r a l . CONCLUSIONS S e l e c t e d - a r e a da rk f i e l d has shown t h a t t h e r e i s an i n t i m a t e r e l a t i o n s h i p between t h e i n o r g a n i c m a t r i x components o f d e n t i n and e n a m e l , and c o n v e n t i o n a l EM o f d e m i n e r a l i z e d s e c t i o n s o f t h e DEJ h a s d e m o n s t r a t e d t h a t t h i s r e l a t i o n s h i p i n c l u d e s t h e o r g a n i c m a t r i x c o m p o n e n t s . These r e s u l t s , combined w i t h t he i n f o r m a t i o n known a b o u t 71 t h e t e m p o r a l e v e n t s o f DEJ f o r m a t i o n , s u g g e s t t h a t t h e f o r m a t i o n o f enamel i s dependent upon d e n t i n m i n e r a l i z a t i o n . I f t h i s i s t h e c a s e , t h e n enamel f o r m a t i o n must be c o n s i d e r e d a s e c o n d a r y m i n e r a l i z a t i o n p r o c e s s t h a t i s i n i t i a t e d by m a t r i x v e s i c l e s w i t h i n p r e d e n t i n . D e n t i n a p a t i t e c r y s t a l s grow e p i t a x i a l l y t o t he s u r f a c e o f d e n t i n and t h e n i n t o t h e enamel m a t r i x where t h e y a r e bounded by t h e enamel s h e a t h and t a k e on t he c h a r a c t e r i s t i c s o f enamel c r y s t a l s ( F i g . 9 d ) . 72 REFERENCES Appleton, J . , and D.C. Morris. 1979. 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U l t r a s t r u c t u r a l l o c a l i z a t i o n o f o s t e o c a l c i n i n r a t t o o t h germs by immunogold s t a i n i n g . H i s t o c h e m i s t r y 8 9 : 5 0 9 - 5 1 4 . E a n e s , E . D . , a n d . A.W. H a i l e r . 1 9 8 5 . L i p o s o m e - m e d i a t e d c a l c i u m p h o s p h a t e f o r m a t i o n i n m e t a s t a b l e s o l u t i o n s . C a l c i f . T i s s u e I n t . 3 7 : 3 9 0 - 3 9 4 . E e c k o u t , Y . , and G. Maes. 1 9 7 7 . F u r t h e r s t u d i e s on t he a c t i v a t i o n o f p r o c o l l a g e n a s e s , t he l a t e n t p r e c u r s o r o f bone c o l l a g e n a s e . E f f e c t o f 74 l y s o s o m a l c a t h e p s i n B, p l a s m i n , k a l l i k r e i n and spon taneous a c t i v a t i o n . B i o c h e m . J . 1 6 6 : 2 1 - 2 8 . E i s e n m a n n , D . R . , and P . L . G l i c k . 1 9 7 2 . U l t r a s t r u c t u r e o f i n i t i a l c r y s t a l f o r m a t i o n i n d e n t i n . J . U l t r a s t r u c t . 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E l e c t r o n m i c r o s c o p i c i n v e s t i g a t i o n o f a m o r p h o l o g i c b a s i s f o r t h e m i n e r a l i z a t i o n p a t t e r n i n r a t i n c i s o r e n a m e l . A n a t . R e c . 1 7 6 : 2 8 9 - 3 0 6 . G l i m c h e r , M . J . 1959 . M o l e c u l a r b i o l o g y o f m i n e r a l i z e d t i s s u e s w i t h p a r t i c u l a r r e f e r e n c e t o bone . R e v . Mod. P h y s . 3 1 : 3 5 9 - 3 9 3 . G l i m c h e r , M . J . , L . C . B o n a r , and E . J . D a n i e l . 1 9 6 1 . The m o l e c u l a r s t r u c t u r e o f t h e p r o t e i n m a t r i x o f b o v i n e d e n t a l e n a m e l . J . M o l . B i o l . 3 : 5 4 1 - 5 4 6 . G r a n s t r o m , G . 1984 . F u r t h e r e v i d e n c e o f an i n t r a v e s i c u l a r Ca2+-pump i n o d o n t o b l a s t s f rom r a t i n c i s o r s . A r c h s . O r a l B i o l . 2 9 : 5 9 9 - 6 0 6 . G r e e n b e r g , G . , P . B r i n g a s , and H . C . S l a v k i n . 1 9 8 3 . 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