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The development of the rat flexor digital tendon, a fine structure study
© 1967 by Academic Press Inc.
354
J. U L T R A S T R U C T U R E R E S E A R C H
18, 354-376 (1967)
The Development of the Rat Flexor Digital Tendon, a Fine Structure Study 1 THEODORE K. GREENLEE, JR., 2 AND RUSSELL ROSS8' 4
Departments of Pathology and Oral Biology, Schools of Medicine and Dentistry, University of Washington, Seattle, Washington 98105 Received June 23, 1966 The development of hind feet synovial sheath flexor tendons has been examined in fetal and postnatal rats. The tendons were first identified as discrete collections of cells in 16-day fetuses. Synovial sheath formation had commenced in 18-day fetuses and was complete by birth. Cell junctions or adhesion sites were noted between the cells that form the tendon proper and between the cells of the visceral synovium. However, no junctions were observed between the cells of these two different populations. The interaction between growth forces and the maintenance of structural integrity by cell adhesions may explain the development of specific structural entities within the tendon. Poorly differentiated mesenchymal cells in 12-day fetuses containing predominantly free cytoplasmic ribosomes differentiate into tendon flbroblasts with a prominent rough-surfaced endoplasmic reticulum and Golgi complex. Besides collagen fibrils in the extracellular space small elastic fibers were also identified and followed in their development. T e n d o n s have been extensively utilized as a relatively p u r e source of collagen f o r chemical a n d physical studies of this protein; however, c o m p a r a t i v e l y little a t t e n t i o n has been p a i d to the cells in tendons. F i t t o n J a c k s o n in her fine structure s t u d y (7) utilized d e v e l o p i n g a v i a n t e n d o n as a m o d e l for examining collagen fibrillogenesis. Fibrillogenesis has also been e x a m i n e d d u r i n g the process of t e n d o n r e g e n e r a t i o n (6, 21, 30). I n the present study the d e v e l o p m e n t of this relatively c o m p l e x biological entity was e x a m i n e d with p a r t i c u l a r a t t e n t i o n to the c o m p l e x cell-to-cell relationships within the t e n d o n a n d the f o r m a t i o n of the t e n d o n sheath. Intercellular c o n t a c t z This investigation was supported in part by U.S. Public Health Service Research Grants nos. DE-01703, HE-03174, GM-13543, and GM-100-08. 2 The work was done while Dr. Greenlee was a Postdoctoral Trainee in Experimental Pathology; his present address is Department of Surgery, Division of Orthopedics, College of Medicine, University of Florida, Gainesville, Florida. 3 Dr. Ross is the holder of a Career Development Award No. DE-9053. Reprint requests should be sent to Dr. Ross.
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sites have l o n g been recognized to be i m p o r t a n t in relation to p h e n o m e n a such as m a i n t e n a n c e of structural integrity in epithelia, c o n t a c t inhibition, a n d t r a n s m i s s i o n of nerve impulses. H o w e v e r , relatively little attention has been p a i d to the role p l a y e d b y cell-to-cell contacts ill connective tissues where they are less a b u n d a n t a n d therefore n o t so obvious (25). This r e p o r t d e m o n s t r a t e s the i m p o r t a n c e of c o n t a c t sites between fibroblasts in the f o r m a t i o n of specific structural entities in developing tendon, including the f o r m a t i o n of the t e n d o n sheath. Changes in c y t o p l a s m i c organelles d u r i n g differentiation of the t e n d o n fibroblasts a n d the d e v e l o p m e n t of collagen a n d elastic fibers in this structure will also be presented.
MATERIALS AND METHODS Tendon development was studied in a series of fetal and postnatal rats by light and electron microscopy. Fetal age was estimated on the basis of the time of conception. F o u r to seven fetuses of approximately similar age were obtained by myotomy of the uterus. Appropriate parts from these and from postnatal animals under anesthesia were removed and prepared for electron microscopy as described below. a. Twelve-day fetal limb buds were fixed in 2% osmium tetroxide (s-collidine buffer) (1), 3 % osmium tetroxide (phosphate buffer), 1.2 % glutaraldehyde (phosphate buffer), postfixed in phosphate-buffered osmium tetroxide, or 6 % glutaraldehyde (phosphate buffer) with 0.1% alcian blue postfixed in phosphate-buffered osmium. b. Sixteen-day fetal limb buds were removed and fixed in 2 % osmium tetroxide (s-collidine buffer). c. Eighteen-day fetal limb buds and separate rays were removed and fixed in s-collidine buffered osmium tetroxide or 1.2% glutaraldehyde with 0.1% alcian blue (3), postfixed in phosphate-buffered osmium tetroxide. d. Newborn and 5-day-old rat digits were removed and fixed in 2 % osmium tetroxide (s-collidine buffer). e. In 30-day-old specimens the distal 2 m m of the digits were removed intact and the deep flexor tendons were dissected from the sheath and placed under tension before fixation in 2 % osmium tetroxide (s-collidine buffer). All fixatives were buffered at p H 7.4. Osmium fixation took place at 4°C, whereas glutaraldehyde fixation was carried out at room temperature. Fixation times varied from 1 to 3 hours depending upon the age of the animals. Tissues were dehydrated in a graded series of alcohols and embedded in epoxy resins (16). The specimens were degassed in a desiccator under vacuum for 3 hours and polymerized at 65°C for 48 hours. Each specimen was oriented on an aluminum chuck to obtain either transverse or longitudinal sections of the tendon. The sections were stained with either (a) 1% aqueous uranyl acetate followed by Millonig's lead (18), (b) 1% aqueous uranyl acetate, (c) Millonig's lead, or (d) 1% aqueous phosphotungstic acid, and examined in an R C A E M U 3 G electron microscope. One-micron sections were stained with azure IX methylene blue (23) or Gomori's aldehyde fuchsin (10) for light microscopy.
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RESULTS
Light microscopic observations Twelve-day fetus. All the m e s e n c h y m a l cells within the confines of the limb b u d were similar in appearance a n d there was no sign of cartilaginous rays or t e n d o n s at this stage of development. Mitotic figures were n u m e r o u s , a n d n o fibrillar extracellular material was evident (Fig. 1).
Thirteen- to sixteen-day fetuses. By the thirteenth day c o n d e n s a t i o n s of cells were seen representing the rays of the foot, b u t n o evidence of t e n d o n s was a p p a r e n t u n t i l the fifteenth or sixteenth day of i n t r a u t e r i n e development. A t the sixteenth fetal day three cellular c o n d e n s a t i o n s could be seen in the fingerlike projections of the limb b u d (Fig. 2). Of these, the central c o n d e n s a t i o n represented the cartilaginous anlage of the b o n y ray; the dorsal crescent-shaped c o n d e n s a t i o n represented the early extensor t e n d o n ; a n d the p l a n t a r c o n d e n s a t i o n , the site of the future flexor t e n d o n . There was n o evidence of sheath f o r m a t i o n at this time, n o r could collagen be identified by light microscopy. Eighteen-day fetus. By the eighteenth day the t e n d o n s were well delineated f r o m the s u r r o u n d i n g mesenchyme by a few rows of circumferential cells that represented the first stage of sheath f o r m a t i o n , b u t n o synovial cavity could be observed (Figs. 3 a n d 10).
Newborn rat. A t birth the flexor digital t e n d o n s h a d a well developed sheath a n d synovial cavity (Fig. 4). Areas of specialization could be seen within the sheath t h a t were destined to become the pulley which prevents " t e n t i n g " of the t e n d o n d u r i n g
FIG. 1. The light micrograph was taken of a 1 # epoxy transverse section of a 12-day fetal limb bud. Both undifferentiated mesenchymal cells (m) and endothelial cells (e) of capillaries are visible. There is no suggestion of cartilage or tendon formation at this stage. Fixed with glutaraldehyde, postfixed in osmium tetroxide. Stained with azure II methylene blue. × 480. FIG. 2. A light micrograph of a transversely sectioned projection from a 16-day fetal limb bud Three cell condensations can be seen. The central condensation will become the cartilaginous ray (c), the crescent-shaped condensation the extensor tendon (et), and the third condensation the flexor tendons (ft). No differentiation between the cells that will form the tendon itself and the synovial sheath can be seen at this stage. Fixed in osmium tetroxide. Stained with azure II methylene blue. × 173. FIG. 3. The transversely sectioned tendons in this micrograph represent the superficial flexor tendon (st) and the deep flexor tendon (dt) of an 18-day fetal digit. The circumferential cells surrounding both tendons constitute the synovial sheath (ss). There is a suggestion of cleft formation between the sheath and the tendon (c) representing the future sheath cavity. In a section taken more distal to the area shown here (Fig. 10) in a region where the superficial tendon has inserted, the tendon appears less well differentiated and the circumferential cells cannot be separated into their component parts. Fixed in glutaraldehyde, postfixed in osmium tetroxide. Stained with azure II methylene blue. x 314. FI~. 4. At birth the cavity of the sheath has formed (sc), as can be seen in this micrograph of a transverse section. In this region the superficial tendon has split (st) and the deep tendon (dt) passes through to its insertion into the distal phalanx. It is apparent that the circumferential cells of the 18-day fetus form the visceral synovia (vs) of the tendon as well as the synovial sheath (ss). Fixed in osmium tetroxide. Stained with toluidine blue. × 237.
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T. K. GREENLEE, JR. AND R. ROSS
flexion of the digits. A layer of cells c o u l d be seen s u r r o u n d i n g the t e n d o n t h a t were o r i e n t e d at right angles to the cells c o m p r i s i n g the t e n d o n itself. This circumferential layer of cells represented the visceral synovium. Collagen fibers were easily identified within the t e n d o n between the cells. B l o o d vessels were n o t o b s e r v e d within the substance of the t e n d o n b u t were present r u n n i n g a l o n g the d o r s a l surface of the deep flexor tendon. O n e - m i c r o n e p o x y sections stained with G o m o r i ' s a l d e h y d e fuchsin d e m o n s t r a t e d structures p r e s u m e d f r o m their a p p e a r a n c e a n d staining characteristics to be elastic fibers. Five- to thirty-day-old rats. F i v e - d a y - o l d t e n d o n s showed n o significant changes f r o m the n e w b o r n s other t h a n an increase in collagen content. T h e t e n d o n s of 30-day-old a n i m a l s a p p e a r e d grossly similar to those seen at 6 m o n t h s of age except for the size of the latter, which was increased. The visceral layer of synovial cells was n o t easily seen because it was d a m a g e d d u r i n g the r e m o v a l of the t e n d o n s f r o m their sheaths, a p r o c e d u r e necessary to o b t a i n a d e q u a t e fixation. W h e r e it c o u l d be examined, the visceral s y n o v i u m a p p e a r e d to consist of a single layer of cells. A s in the n e w b o r n animal, small a l d e h y d e fuchsin positive fibers c o u l d be seen a n d the a m o u n t of collagen was greatly increased.
Electron microscopic observations Twelve-day fetus. C o n s i d e r a b l e difficulty was e n c o u n t e r e d in o b t a i n i n g a d e q u a t e cellular fixation at this age. D i s r u p t e d p l a s m a m e m b r a n e s were seen in the 12-day fetus following fixation with s-collidine buffered o s m i u m a l t h o u g h this fixative p r o v e d satisfactory for the m a j o r i t y of the later stages of d e v e l o p m e n t . G l u t a r a l d e h y d e c o n t a i n i n g alcian blue (3) followed b y o s m i u m tetroxide p r o v i d e d i m p r o v e d fixation t h o u g h o c c a s i o n a l m e m b r a n e b r e a k s c o u l d still be found. The extracellular spaces were d e v o i d of fibrillar m a t e r i a l a n d only two cell types
FIG. 5. This electron micrograph shows some of the relatively undifferentiated mesenchymal cells in the 12-day fetal limb bud. Junction sites (is) between two of the cells are apparent. Nuclei (n), mitochondria (m), and numerous aggregates of free ribosomes (fr) are seen. Only occasional lamellae of rough endoplasmic reticulum (er) and Golgi vesicles (g) can be found at this age. An invagination of cytoplasm can be seen in one of the nuclei (arrow). Though the fixation here is improved over s-collidine buffered osmium, occasional breaks in the cell membranes can still be found. Fixed with glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 17,500. Fie. 6. A higher magnification of a junctional site similar to those seen in Fig. 5. The membranes are separated by approximately 200 A at these two sites (is). The cytoplasm adjacent to the junctional site appears increased in density. Clusters of free ribosomes (fr) and some membrane-bound ribosomes (er) are also seen in this micrograph. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 53,400.
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T. K. GREENLEE, JR. AND R. ROSS
w e r e seen b e n e a t h the e p i t h e l i u m of the l i m b b u d . T h e s e c o n s i s t e d of c a p i l l a r y e n d o t h e l i u m a n d w h a t a p p e a r e d to be i m m a t u r e m e s e n c h y m a l cells (Fig. 5). F o c a l j u n c t i o n a l sites, similar in a p p e a r a n c e to t h e i n t e r m e d i a t e j u n c t i o n s d e s c r i b e d in e p i t h e l i u m (4), w e r e o b s e r v e d b e t w e e n these m e s e n c h y m a l cells (25). I n t h e s e r e g i o n s the cell m e m b r a n e s
w e r e n e a r l y p a r a l l e l a n d s e p a r a t e d by a d i s t a n c e o f
a p p r o x i m a t e l y 200 •. B o t h the i n t e r v e n i n g space a n d the c y t o p l a s m a d j a c e n t to t h e j u n c t i o n a l sites w e r e i n c r e a s e d in d e n s i t y (Fig. 6). A t this e a r l y stage of d e v e l o p m e n t the n u c l e u s o c c u p i e d t h e m a j o r p a r t of e a c h of t h e s e cells. T h e c y t o p l a s m c o n t a i n e d m i t o c h o n d r i a , a few c i s t e r n a e of r o u g h e n d o p l a s m i c r e t i c u l u m a n d i s o l a t e d G o l g i vesicles. M o s t of the c y t o p l a s m h o w e v e r w a s o c c u p i e d b y n u m e r o u s clusters of free r i b o s o m e s (Figs. 5 a n d 6).
FIG. 7. This electron micrograph demonstrates tendon cells in a longitudinal section of a 16-day fetus. Collagen fibrils (co) can be identified in the extracellular space by their characteristic banding. A second population of unbanded fibrils (f) (100 ~ diameter) can also be seen. Rough endoplasmic reticulum (er) and Golgi complex (g) are more abundant at this stage than in the mesenchymal cells of the 12-day fetus (Fig. 5), but clusters of free ribosomes (fr) are still prominent. The cell nucleus (n) and mitochondria (m) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 18,200. FrG. 8. In an electron micrograph of a transversely sectioned 16-day fetal tendon, 100 /~ fibrils 0 c) of young elastic fibers are seen in the extracellular space. An obliquely sectioned junction site (is) between two of the cells can be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 16,000. FIG. 9. This low power electron micrograph of a transverse section from an 18-day fetal tendon demonstrates the area of junction between the tendon proper (Tendon) and the circumferentially oriented cells surrounding it. Fig. 10 is a light micrograph of a 1 t~ section taken adjacent to the thin section seen in Fig. 9. The circumferential cells closely applied to the tendon will become part of the visceral synovium (Vis. Syn.) and the cells at the top of the picture accompanied by longitudinally oriented collagen fibrils will become the fibrous sheath (Fib. Sheath). The sheath cavity will form in the area lying between these two regions. The cells in this region will segregate. Some of them will become the parietal synovium, and the others will remain as the visceral synovial layer of the tendon proper. No clear line of cleavage between these two can be seen at this stage. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 4500. FIG. 10. This light micrograph of a 1 # section taken after the thin section used for Fig. 9 shows the tendon surrounded by the circumferentially oriented cells (cc) that will form the synovial sheath and visceral synovial layer of the tendon (Ten.). Fixed in osmium tetroxide. Stained with azure II methylene blue. x 276. FIG. 11. There has been a marked change in the depth of the tendon in the 18-day fetus, as compared, to earlier stages, as seen in this electron micrograph of a transverse section. The increase in numbers and diameter of collagen fibrils (co) has caused the cells to separate and maintain contact at junctional sites found at the end of long cytoplasmic processes. Numerous collections of 100 A fibrils representing early elastic fiber (el) formation can also be seen. The rough endoplasmic reticulum and Golgi complex are more prominent in the tendon fibroblasts at this time. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 5700. FIG. 12. This micrograph demonstrates part of the tendon proper from and 18-day fetus similar to that seen in Fig. 11 demonstrating that in longitudinal section the cells are more spindle shaped than those seen in Fig. 7. The amount of collagen (co) is also increased. The contents of the cisternae of rough endoplasmic reticulum (er) are denser than the remainder of the cytoplasm, and free ribosomal clusters may still be observed. The nucleus (n) and mitochondria (m) can also be identified. Fixed in glutaraldehyde containing alcian blue. Stained with uranyl acetate and lead. x 13,500.
8
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T . K . GREENLEE~ JR. AND R. ROSS
Sixteen-day fetus. The 16-day fetal tendon anlage did not contain a layer of circumferential cells as was found at the later stages. Collagen fibrils of approximately 200 A diameter were seen in the extracellular spaces between the cells of the tendon. A second population of fibrils approximately 100 ]t in diameter that did not display the characteristic banding of collagen was also present (Figs. 7 and 8). These fibrils were presumed to represent the precursors of elastic fibers subsequently identified in the older animals (11). The fibroblasts of the tendon contained many more profiles of rough endoplasmic reticulum and Golgi regions than did the less mature cells on the twelfth day of development. However, many free ribosomal clusters were still present within these cells and junctional sites were seen between adjoining fibroblasts (Fig. 8). Eighteen-day-fetus. After 18 days of fetal development the forming tendon was surrounded by a layer of circumferentially oriented cells 8-10 cells thick. At a later stage of development these cells will separate and be segregated into three separate structures. The innermost cells will remain with the tendon to become its visceral synovium. The outermost will become the fibrous sheath, and the cells remaining between will segregate at the time of cavity formation to produce the parietal synovium. No clear separation between these layers was apparent at this stage (Figs. 9 and 10). As in the tendon proper, there are junctional regions between these circumferentially oriented cells, but not between the sheath cells and the fibroblasts in the mass of the tendon. The amount of collagen between the cells by this time had increased, forcing the cells farther apart. The fibroblasts retain their intercellular contacts through junctional sites located at the ends of relatively slender cell processes (Figs. 11 and 13). At this stage the collagen fibrils were approximately 250 A in diameter and the second population of extracellular fibrils (100 A diameter) formed larger aggregates than those seen in the 16-day-old fetuses (Fig. 14). No qualitative changes were apparent in cell organelles, but there appeared to be a relative increase in the amount of rough-surfaced endoplasmic reticulum and a decrease in the number of clusters of free cytoplasmic ribosomes (Figs. 11 and 12). Newborn. As stated previously, the tendon sheath cavity was present at birth (Figs. 4 and 15). The visceral synovium consists of circumferentially oriented cells and collagen fibrils (Fig. 15). At this age, tendon cells appeared to be more compressed owing to the increase in extracellular fibrils. Cell-to-cell contact, however, was maintained by thin cytoplasmic processes attached by junctional sites similar to those previously described (Fig. 16). Concomitant with the increase in extracellular material, the fibroblasts appeared fusiform in longitudinal section in contrast to their appearance at the earlier stages of formation (Fig. 17). Two changes could be seen in the extracellular connective tissue elements at this
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Eio. 13. This micrograph demonstrates a higher magnification of one of the intercellular attachment sites from an 18-day fetal tendon. The membranes are separated by approximately 200 ~, and the cytoplasm is denser adjacent to these sites. Fixed in glutaraldehyde containing alcian blue. Stained with uranyl acetate and lead. x 79,100. FI~. 14. In this micrograph from an 18-day fetus two transversely sectioned early elastic fibers (ela and elb) are shown. The 100 ~ fibrils (f) which constitute the major component of the immature elastic fiber are tubular in profile. Elastic fiber a (ela) is made up solely of 100 N fibrils while elastic fiber b (elb) contains a few small amorphous central areas (ca) surrounded by 100 N fibrils. Collagen fibils (co) of approximately 250 N diameter are present in the extracellular space. The collagen fibril (arrow) surrounded by a membrane is in the extracellular space. The relatively dark staining of the collagen fibrils and central areas of the elastic fibers seen here compared with those in Figs. 16 and 22 is characteristic of tissue subjected to glutaraldehyde fixation as compared with primary osmium fixation. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 89,500.
time: (a) The collagen fibrils were increased in diameter (430 A) a n d were relatively u n i f o r m in size. The 100 ~ fibrils that were observed from the sixteenth day of fetal life appeared to be part of a more complex structure at the time of birth. These structures consist of central a m o r p h o u s cores that stain poorly with u r a n y l acetate a n d / o r lead s u r r o u n d e d by the 100 ~ fibrils. The latter are t u b u l a r in cross section a n d are beaded in l o n g i t u d i n a l section (Figs. 16 a n d 17). The aggregates of these two c o m p o n e n t s can be identified as elastic fibers as a result of their correspondence 2 4 - - 6 7 1 8 2 3 J . Ultrastructure Y~esearch
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T . K . GREENLEE, JR. AND R. ROSS
to s t r u c t u r e s w h i c h stain w i t h a l d e h y d e f u c h s i n in l i g h t m i c r o s c o p i c p r e p a r a t i o n s a n d t h e i r s i m i l a r i t y to elastic fibers seen in b o v i n e f e t a l l i g a m e n t u m n u c h a e (11). I t w a s f o u n d t h a t w h e n sections w e r e s t a i n e d w i t h u r a n y l acetate, M i l l o n i g ' s lead, o r p h o s p h o t u n g s t i c acid, c h a r a c t e r i s t i c s t a i n i n g r e a c t i o n s w e r e n o t e d f o r t h e t w o c o m p o n e n t s of the elastic fiber as well as f o r t h e c o l l a g e n fibrils. T h e 100 A fibrils s t a i n e d w i t h l e a d a n d / o r a q u e o u s u r a n y l a c e t a t e b u t n o t w i t h p h o s p h o t u n g s t i c acid, w h e r e a s t h e central core regions stained with phosphotungstic acid but not with lead or uranyl acetate. I n c o n t r a s t , t h e c o l l a g e n fibrils s t a i n e d w i t h u r a n y l a c e t a t e a n d p h o s p h o t u n g stic a c i d b u t n o t w i t h lead. Rough
e n d o p l a s m i c r e t i c u l u m a n d G o l g i vesicles w e r e m o r e p r o m i n e n t in t h e
t e n d o n f i b r o b l a s t s at this age t h a n in t h e 18 d a y fetuses, a n d free r i b o s o m a l a g g r e -
FIG. 15. This electron micrograph demonstrates the relationship between the tendon proper and the visceral synovium in the newborn animal as seen in transverse section. The collagen fibrils (cot) and the cells of the tendon proper (Ten. C) are transversely sectioned while the synovial cells (Syn. C) and their accompanying fine collagen fibrils (cos) are longitudinally oriented. The sheath cavity (Car.) is superficial to the synovial cells. Junction sites (ys) can be seen between the cells of the tendon, but not between the tendon cells and the synovial cells. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 12,400. Fla. 16. Two cells in this transverse section of newborn tendon still maintain contact by fine cytoplasmic processes. Junction sites (is) similar to those seen earlier are present. Both collagen fibrils (co) and elastic fibers (el) are found in the extracellular space. The rough endoplasmic reticulum (er) and Golgi Complex (g) are more prominent. Inset shows a higher magnification of one of the elastic fibers. This fiber consists of tubular fibrils (f) (100 A in diameter) and central cores (ca) that are almost amorphous in appearance. The elastic fiber in this micrograph is found in a cytoplasmic niche, a frequent finding in the tendon. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 21,400. Inset × 43,700. FIG. 17. When an elastic fiber from a newborn such as that seen in Fig. 16 is sectioned longitudinally, it appears as shown in this micrograph. The 100 A fibrils (f) do not contain apparent crossbanding as do the collagen fibrils (co) but they do appear to be beaded. The central core areas (ca) seen in cross section now appear as streaks. The elastic fiber shown here is adjacent to the cell membrane and runs a straighter course than do the collagen fibrils in the micrograph. The ergastoplasm (er) and Golgi complex (g) are prominent in the fibroblast. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 22,400. FIG. 18. This electron micrograph of a transverse section of a 30-day-old rat tendon demonstrates the marked increase in collagen (co). The cells are farther apart, but fine cytoplasmic processes (p) course between the bundles of fibrils and maintain cell contact. In some regions a mantle of fine filaments (fi) now separate the plasma membranes of the cells from the collagen. Abundant rough endoplasmic reticulum (er) and Golgi complex (g) are seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 15,000. FIG. 19. In this micrograph of a longitudinal section of a 30-day-old rat tendon, the cells tend to lie in rows separated by large bundles of collagen fibrils (co). Between adjacent cells the extracellular space is filled with fine filaments (fi). At this age the cytoplasm of the cells is packed with lamellae of rough endoplasmic reticulum (er) and Golgi complex (g). Mitochondria (m) are present, and a lipid droplet (l) can be seen in one of the cells. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 15,000. FIG. 20. This is a higher magnification of two adjacent cell membranes from a longitudinal section of a 30-day-old rat tendon. The numerous peripheral vesicles (v) were not a prominent feature in the earlier specimens. Fine filaments (fi) are prominent between the cells, and the rough endoplasmic reticulum (er) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 42,500.
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T . K . GREENLEE, JR. AND R. ROSS
gates, although diminished in number, were still present. Peripheral vesicles were occasionally seen in these cells as well. Thirty-day-old animals. At this stage the cellularity of the tendon was decreased in association with the increase in amount of extracellular collagen. Many fine cytoplasmic processes could be seen to course obliquely between the bundles of collagen. Presumably their function is to maintain cell contact (Fig. 18). The amount of rough endoplasmic reticulum and Golgi components had increased as compared with the newborn, and few, if any, free ribosomal aggregates were seen. Occasional lipid droplets and numerous peripheral vesicles were also present in these cells (Figs. 19, 20, and 22). A finding not previously observed was the presence of a mantle of fine extracellular filaments between adjacent tendon cells and between the plasma membranes of fibroblasts and the collagen fibrils (Figs. 12, 20, and 22). The collagen fibrils had continued to increase in size although the range of diameters (800-1600 A) was less homogeneous than that observed during the earlier stages of development (Fig. 22). In contrast, the elastic fibers had not changed in overall diameter (about 2500 ~ ) although the area of each fiber occupied by the central core regions had increased (Figs. 21 and 22). DISCUSSION The adult synovial sheath tendon contains three components that together constitute the functional tendon. These are: (a) the tendon proper, (b) the visceral synovium adherent to the tendon proper, and (c) the parietal synovium and sheath. Such a tendon is derived from a homogeneous population of cells that in the 12-day fetus appears to consist of relatively undifferentiated mesenchymal cells. During development, these cells differentiate into the three distinct populations that will form these three structures, all of which can be identified by birth.
Intercellular relationships Specific sites of cell attachment can be seen between the connective tissue cells within each of these three structures. Yet no sites of cell attachment were observed between the cells of any two neighboring structures, such as between the tendon proper and the visceral synovium, or between the cells of the visceral synovium and those in the parietal synovium. Thus, it can be concluded that the presence of contact sites plays a role in the maintenance of structural integrity within each of these components. As tendon motion begins, the parietal and visceral synovial cells are segregated, resulting in a sp.ace between the visceral and parietal synovia. A similar conclusion
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FIG. 21. Elastic fibers are difficult to find in longitudinal sections of the 30-day-old rat tendon. This micrograph shows two obliquely sectioned fibers. The amorphous central areas (ca) are larger and more prominent at this age than in the newborn seen in Fig. 17. The 100/k fibrils (f) are still present and are unbanded as compared to the collagen (co). Note the cell process (p) adjacent to the elastic fibers. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 16,000. FIG. 22. If elastic fibers such as those seen in Fig. 21 are transversely sectioned, they appear as seen in this micrograph. The central core areas (ca) of these two fibers take up a larger percentage of the fiber than in Fig. 16, and the 100/~ fibrils (f) are less prominent. Elastic fibers remain in close proximity to the plasma membrane of the cells. Collagen fibrils (co) are less homogeneous in size than in the newborn and are greatly increased in diameter (800-1600/~). The cell nucleus (n), rough endoplasmic reticulum (er), and peripheral vesicles (v) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 34,000.
was r e a c h e d b y Shields
(27) in a n e a r l y light m i c r o s c o p e s t u d y of d e v e l o p i n g pig
s y n o v i a l t e n d o n s . S y n o v i a l c a v i t y f o r m a t i o n a p p e a r s to o c c u r in a s i m i l a r f a s h i o n in t h e d e v e l o p m e n t of s y n o v i a l j o i n t s in c h i c k e m b r y o s
(14).
Forces acting upon the tendon during development It m a y be t h a t the o r i e n t a t i o n of t h e cells in e a c h of t h e c o m p o n e n t s of the t e n d o n is a f f e c t e d b y t h e f o r c e t h a t is a p p l i e d to it a n d is t r a n s m i t t e d i n t e r c e l l u l a r l y v i a the cell a t t a c h m e n t sites. T h e t e n d o n p r o p e r is a t t a c h e d to b o t h b o n e a n d m u s c l e . T h e r e -
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T . K . GREENLEE, JR. AND R. ROSS
fore, the forces resulting from bone growth and muscle contraction would be transmitted from cell to cell in a longitudinal direction and would result in cells and extracellular fibrils oriented parallel to the developing bony rays of the foot. In contrast, the visceral synovial cells surround the tendon and contain no attachments to either the tendon proper, the bone, or the muscle. The forces acting upon this group of cells would result from the increase in girth of the tendon and would be approximately at a right angle to the longitudinal forces acting upon the tendon proper, resulting in a circumferentially oriented structure. The parietal synovium and sheath together form a horseshoe-shaped structure that is attached along the bones of the ray and surrounds the tendon. As with the visceral synovium, the primary force affecting the orientation of these cells will be the increasing girth of the tendon during development.
Cell differentiation During the period of tendon growth and maturation from the 12-day embryo to birth, several changes occur in the number and distribution of organelles within the fibroblasts of the tendon proper. These consist of (a) a decrease in the number of free aggregates or ribosomes; (b) an increase in the amount and distribution of rough endoplasmic reticulum (the cisternal membranes of the mature fibroblast characteristically contain large aggregates of attached ribosomes, often appearing as double rows or in a spiral formation), (c) an increase in the Golgi complex; and (d) an increase in the amount of peripheral vesicles. These changes are similar to those described by Goldberg and Green (9) in tissue culture fibroblasts during the transition from a proliferative phase of growth, when relatively little collagen was synthesized, to a stationary growth phase when collagen synthesis was actively taking place. The poorly differentiated mesenchymal cells in the 12-day embryo are similar in appearance to the early cells described by Hay (12, 13) and Salpeter (26) in the regenerating amphibian limb bud, as well as to poorly differentiated cells studied in other tissues (2, 15, 17, 19, 29). As in other investigations, it would appear that the development of an extensive rough endoplasmic reticulum can be correlated with the enhanced ability of these fibroblasts to synthesize and secrete the extracelhilar substances of the connective tissues of the tendon, i.e., collagen, elastin, and various protein polysaccharide complexes (5, 8, 20, 22, 24, 28). The role played by the numerous peripheral vesicles, particularly preminent near the plasma membrane of the fibroblasts in 30-day-old animals, is not clear. It is possible that they may be related to either pinocytosis or the secretion of extracellular materials; however, no evidence in this regard can be provided by this study.
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The extracellular components Collagen fibrils. Collagen fibrils were present between the cells of the flexor tendons in 15- and 16-day fetuses. These fibrils were approximately 160 • in diameter. By 18 days of fetal life, the fibrils had increased in quantity and were between 200 and 250 A in diameter. A further increase in fibril diameter to approximately 400 A was observed at birth. At one month of age fibril diameters had become less homogeneous in the tendon and had increased markedly in size (800-1600 ,~ diameter). Similar age changes were demonstrated by Fitton Jackson (7) in her study of developing chick tendons. Elastic fibers. Of interest in this study was the presence of elastic fibers that reached a m a x i m u m diameter of approximately 2500 A. These fibers consisted of central a m o r p h o u s cores surrounded by 100 /~ tubular fibrils. The fine structure of these elastic fibers were described in detail in a previous publication (11). The function of these fibers within an inelastic structure such as a tendon is not clear. In the spinal ligaments and the connective tissues of arteries, elastic fibers predominate, whereas in tendon collagen fibrils are the major structural component. It is possible that the relatively small number of elastic fibers in tendon may play a role in the maintenance of a rest relationship between collagen fibrils following vigorous muscular contraction. Relatively little attention has been paid to this interesting aspect of tendon structure.
The authors would like to acknowledge the technical assistance of Dawn Bockus, Leslie Caldwell, Rene Collman, Ingrid Klock, and Franque Remington in the preparation of the thin sections; the photographic assistance of Johsel Namkung in preparation of the micrographs for publication; Sam Lewis for his help in providing the fetal rats; the secretarial services of Mrs. Dorris Knibb; and the editorial services of Miss Alison Ross. The authors are particularly indebted to Drs. Earl P. Benditt and John H. Luft for their help and encouragement during the progress of this study. REFERENCES
1. BENNETT,H. S. and LUFT, J. H., J. Biophys. Biochem. Cytol. 6, 113 (1959). 2. BEHNKE, O. and MOE, M., J. Cell Biol. 22, 633 (1964). 3. COMBS,J. W., Y. Cell Biol. 31, 563 (1966). 4. FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). 5. FEWER,D., THREADGOLD,J. and SHELDON,H., J. Ultrastruct. Res. 11, 166 (1964). 6. FERNANDO,N. V. and MOVAT, H. Z., Lab. Invest. 12, 214 (1964). 7. F~TTONJACKSON,S., Proe. Roy. Soc. B144, 556 (1956). 8. GODMAN,G. C. and LANE, M., J. Cell Biol. 21, 353 (1964). 9. GOLDBERG,B. and GREEN, H., J. Cell Biol. 22, 227 (1964). 10. GOMORI,G., Am. J. Clin. Pathol. 20, 665 (1950). 11. GREENLEE,T. K., JR., ROSS, R. and HARTMAN,J. H., J. CelIBiol. 30, 59 (1966).
376 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
T.K. GREENLEE, JR. AND R. ROSS HAY, E., or. Biophys. Biochem. Cytol. 4, 583 (1958). Develop. Biol. 1, 555 (1959). -HENRIKSON,R. C. and COHEN, A. S., J. Ultrastruct. Res. 13, 129 (1965). HOWATSON,A. F. and HAM, A. W., Cancer Res. 15, 62 (1955). LUFT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). MARK, P. A., RIFKIND, R. A., and DANON, D., Proc. Natl. Acad. Sci. U.S. 50, 336 (1963). MmLON~G,G., J. Biophys. Biochem. Cytol. 11, 736 (1961). PALADE, G. E., J. Biophys. Biochem. Cytol. 1, 58 (1955). PALADE, G. E. and SmKEVITZ,P., J. Biophys. Biochem. Cytol. 2, 671 (1956). PEACH, R., WILLIAMS,G. and CHAPMAN,J. A., Am. J. Pathol. 38, 495 (1961). REVEL,J. P. and HAY, E., Z. Zellforsch. Mikroskop. Anat. 61, 110 (1963). RICHARDSON,K. C., JARRETT,L., and FINKE, E. H., Stain Technol. 35, 313 (1965). Ross, R. and BENDITT,E. P., J. Cell Biol. 27, 83 (1965). Ross, R. and GREENLEE,T. K., JR., Science, 153, 997 (1966). SALPETER,M. M. and SINGER, M., Proc. 5th Intern. Congr. Electron Microscopy, Philadelphia, 1962 Vol. 2, OO. Academic Press, New York, 1962. SHIELDS,R. T., Contrib. Embryol. 15, 53 (1923). SIEKEVITZ,P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 7, 619 (1960). SLAUTTERBACK,D. B. and FAWCETT, D. M.. J. Biophys. Biochem. Cytol. 5, 441 (1959). WASSERMAN,F., Am. J. Anat. 94, 399 (1954).
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18, 354-376 (1967)
The Development of the Rat Flexor Digital Tendon, a Fine Structure Study 1 THEODORE K. GREENLEE, JR., 2 AND RUSSELL ROSS8' 4
Departments of Pathology and Oral Biology, Schools of Medicine and Dentistry, University of Washington, Seattle, Washington 98105 Received June 23, 1966 The development of hind feet synovial sheath flexor tendons has been examined in fetal and postnatal rats. The tendons were first identified as discrete collections of cells in 16-day fetuses. Synovial sheath formation had commenced in 18-day fetuses and was complete by birth. Cell junctions or adhesion sites were noted between the cells that form the tendon proper and between the cells of the visceral synovium. However, no junctions were observed between the cells of these two different populations. The interaction between growth forces and the maintenance of structural integrity by cell adhesions may explain the development of specific structural entities within the tendon. Poorly differentiated mesenchymal cells in 12-day fetuses containing predominantly free cytoplasmic ribosomes differentiate into tendon flbroblasts with a prominent rough-surfaced endoplasmic reticulum and Golgi complex. Besides collagen fibrils in the extracellular space small elastic fibers were also identified and followed in their development. T e n d o n s have been extensively utilized as a relatively p u r e source of collagen f o r chemical a n d physical studies of this protein; however, c o m p a r a t i v e l y little a t t e n t i o n has been p a i d to the cells in tendons. F i t t o n J a c k s o n in her fine structure s t u d y (7) utilized d e v e l o p i n g a v i a n t e n d o n as a m o d e l for examining collagen fibrillogenesis. Fibrillogenesis has also been e x a m i n e d d u r i n g the process of t e n d o n r e g e n e r a t i o n (6, 21, 30). I n the present study the d e v e l o p m e n t of this relatively c o m p l e x biological entity was e x a m i n e d with p a r t i c u l a r a t t e n t i o n to the c o m p l e x cell-to-cell relationships within the t e n d o n a n d the f o r m a t i o n of the t e n d o n sheath. Intercellular c o n t a c t z This investigation was supported in part by U.S. Public Health Service Research Grants nos. DE-01703, HE-03174, GM-13543, and GM-100-08. 2 The work was done while Dr. Greenlee was a Postdoctoral Trainee in Experimental Pathology; his present address is Department of Surgery, Division of Orthopedics, College of Medicine, University of Florida, Gainesville, Florida. 3 Dr. Ross is the holder of a Career Development Award No. DE-9053. Reprint requests should be sent to Dr. Ross.
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sites have l o n g been recognized to be i m p o r t a n t in relation to p h e n o m e n a such as m a i n t e n a n c e of structural integrity in epithelia, c o n t a c t inhibition, a n d t r a n s m i s s i o n of nerve impulses. H o w e v e r , relatively little attention has been p a i d to the role p l a y e d b y cell-to-cell contacts ill connective tissues where they are less a b u n d a n t a n d therefore n o t so obvious (25). This r e p o r t d e m o n s t r a t e s the i m p o r t a n c e of c o n t a c t sites between fibroblasts in the f o r m a t i o n of specific structural entities in developing tendon, including the f o r m a t i o n of the t e n d o n sheath. Changes in c y t o p l a s m i c organelles d u r i n g differentiation of the t e n d o n fibroblasts a n d the d e v e l o p m e n t of collagen a n d elastic fibers in this structure will also be presented.
MATERIALS AND METHODS Tendon development was studied in a series of fetal and postnatal rats by light and electron microscopy. Fetal age was estimated on the basis of the time of conception. F o u r to seven fetuses of approximately similar age were obtained by myotomy of the uterus. Appropriate parts from these and from postnatal animals under anesthesia were removed and prepared for electron microscopy as described below. a. Twelve-day fetal limb buds were fixed in 2% osmium tetroxide (s-collidine buffer) (1), 3 % osmium tetroxide (phosphate buffer), 1.2 % glutaraldehyde (phosphate buffer), postfixed in phosphate-buffered osmium tetroxide, or 6 % glutaraldehyde (phosphate buffer) with 0.1% alcian blue postfixed in phosphate-buffered osmium. b. Sixteen-day fetal limb buds were removed and fixed in 2 % osmium tetroxide (s-collidine buffer). c. Eighteen-day fetal limb buds and separate rays were removed and fixed in s-collidine buffered osmium tetroxide or 1.2% glutaraldehyde with 0.1% alcian blue (3), postfixed in phosphate-buffered osmium tetroxide. d. Newborn and 5-day-old rat digits were removed and fixed in 2 % osmium tetroxide (s-collidine buffer). e. In 30-day-old specimens the distal 2 m m of the digits were removed intact and the deep flexor tendons were dissected from the sheath and placed under tension before fixation in 2 % osmium tetroxide (s-collidine buffer). All fixatives were buffered at p H 7.4. Osmium fixation took place at 4°C, whereas glutaraldehyde fixation was carried out at room temperature. Fixation times varied from 1 to 3 hours depending upon the age of the animals. Tissues were dehydrated in a graded series of alcohols and embedded in epoxy resins (16). The specimens were degassed in a desiccator under vacuum for 3 hours and polymerized at 65°C for 48 hours. Each specimen was oriented on an aluminum chuck to obtain either transverse or longitudinal sections of the tendon. The sections were stained with either (a) 1% aqueous uranyl acetate followed by Millonig's lead (18), (b) 1% aqueous uranyl acetate, (c) Millonig's lead, or (d) 1% aqueous phosphotungstic acid, and examined in an R C A E M U 3 G electron microscope. One-micron sections were stained with azure IX methylene blue (23) or Gomori's aldehyde fuchsin (10) for light microscopy.
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RESULTS
Light microscopic observations Twelve-day fetus. All the m e s e n c h y m a l cells within the confines of the limb b u d were similar in appearance a n d there was no sign of cartilaginous rays or t e n d o n s at this stage of development. Mitotic figures were n u m e r o u s , a n d n o fibrillar extracellular material was evident (Fig. 1).
Thirteen- to sixteen-day fetuses. By the thirteenth day c o n d e n s a t i o n s of cells were seen representing the rays of the foot, b u t n o evidence of t e n d o n s was a p p a r e n t u n t i l the fifteenth or sixteenth day of i n t r a u t e r i n e development. A t the sixteenth fetal day three cellular c o n d e n s a t i o n s could be seen in the fingerlike projections of the limb b u d (Fig. 2). Of these, the central c o n d e n s a t i o n represented the cartilaginous anlage of the b o n y ray; the dorsal crescent-shaped c o n d e n s a t i o n represented the early extensor t e n d o n ; a n d the p l a n t a r c o n d e n s a t i o n , the site of the future flexor t e n d o n . There was n o evidence of sheath f o r m a t i o n at this time, n o r could collagen be identified by light microscopy. Eighteen-day fetus. By the eighteenth day the t e n d o n s were well delineated f r o m the s u r r o u n d i n g mesenchyme by a few rows of circumferential cells that represented the first stage of sheath f o r m a t i o n , b u t n o synovial cavity could be observed (Figs. 3 a n d 10).
Newborn rat. A t birth the flexor digital t e n d o n s h a d a well developed sheath a n d synovial cavity (Fig. 4). Areas of specialization could be seen within the sheath t h a t were destined to become the pulley which prevents " t e n t i n g " of the t e n d o n d u r i n g
FIG. 1. The light micrograph was taken of a 1 # epoxy transverse section of a 12-day fetal limb bud. Both undifferentiated mesenchymal cells (m) and endothelial cells (e) of capillaries are visible. There is no suggestion of cartilage or tendon formation at this stage. Fixed with glutaraldehyde, postfixed in osmium tetroxide. Stained with azure II methylene blue. × 480. FIG. 2. A light micrograph of a transversely sectioned projection from a 16-day fetal limb bud Three cell condensations can be seen. The central condensation will become the cartilaginous ray (c), the crescent-shaped condensation the extensor tendon (et), and the third condensation the flexor tendons (ft). No differentiation between the cells that will form the tendon itself and the synovial sheath can be seen at this stage. Fixed in osmium tetroxide. Stained with azure II methylene blue. × 173. FIG. 3. The transversely sectioned tendons in this micrograph represent the superficial flexor tendon (st) and the deep flexor tendon (dt) of an 18-day fetal digit. The circumferential cells surrounding both tendons constitute the synovial sheath (ss). There is a suggestion of cleft formation between the sheath and the tendon (c) representing the future sheath cavity. In a section taken more distal to the area shown here (Fig. 10) in a region where the superficial tendon has inserted, the tendon appears less well differentiated and the circumferential cells cannot be separated into their component parts. Fixed in glutaraldehyde, postfixed in osmium tetroxide. Stained with azure II methylene blue. x 314. FI~. 4. At birth the cavity of the sheath has formed (sc), as can be seen in this micrograph of a transverse section. In this region the superficial tendon has split (st) and the deep tendon (dt) passes through to its insertion into the distal phalanx. It is apparent that the circumferential cells of the 18-day fetus form the visceral synovia (vs) of the tendon as well as the synovial sheath (ss). Fixed in osmium tetroxide. Stained with toluidine blue. × 237.
~j
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T. K. GREENLEE, JR. AND R. ROSS
flexion of the digits. A layer of cells c o u l d be seen s u r r o u n d i n g the t e n d o n t h a t were o r i e n t e d at right angles to the cells c o m p r i s i n g the t e n d o n itself. This circumferential layer of cells represented the visceral synovium. Collagen fibers were easily identified within the t e n d o n between the cells. B l o o d vessels were n o t o b s e r v e d within the substance of the t e n d o n b u t were present r u n n i n g a l o n g the d o r s a l surface of the deep flexor tendon. O n e - m i c r o n e p o x y sections stained with G o m o r i ' s a l d e h y d e fuchsin d e m o n s t r a t e d structures p r e s u m e d f r o m their a p p e a r a n c e a n d staining characteristics to be elastic fibers. Five- to thirty-day-old rats. F i v e - d a y - o l d t e n d o n s showed n o significant changes f r o m the n e w b o r n s other t h a n an increase in collagen content. T h e t e n d o n s of 30-day-old a n i m a l s a p p e a r e d grossly similar to those seen at 6 m o n t h s of age except for the size of the latter, which was increased. The visceral layer of synovial cells was n o t easily seen because it was d a m a g e d d u r i n g the r e m o v a l of the t e n d o n s f r o m their sheaths, a p r o c e d u r e necessary to o b t a i n a d e q u a t e fixation. W h e r e it c o u l d be examined, the visceral s y n o v i u m a p p e a r e d to consist of a single layer of cells. A s in the n e w b o r n animal, small a l d e h y d e fuchsin positive fibers c o u l d be seen a n d the a m o u n t of collagen was greatly increased.
Electron microscopic observations Twelve-day fetus. C o n s i d e r a b l e difficulty was e n c o u n t e r e d in o b t a i n i n g a d e q u a t e cellular fixation at this age. D i s r u p t e d p l a s m a m e m b r a n e s were seen in the 12-day fetus following fixation with s-collidine buffered o s m i u m a l t h o u g h this fixative p r o v e d satisfactory for the m a j o r i t y of the later stages of d e v e l o p m e n t . G l u t a r a l d e h y d e c o n t a i n i n g alcian blue (3) followed b y o s m i u m tetroxide p r o v i d e d i m p r o v e d fixation t h o u g h o c c a s i o n a l m e m b r a n e b r e a k s c o u l d still be found. The extracellular spaces were d e v o i d of fibrillar m a t e r i a l a n d only two cell types
FIG. 5. This electron micrograph shows some of the relatively undifferentiated mesenchymal cells in the 12-day fetal limb bud. Junction sites (is) between two of the cells are apparent. Nuclei (n), mitochondria (m), and numerous aggregates of free ribosomes (fr) are seen. Only occasional lamellae of rough endoplasmic reticulum (er) and Golgi vesicles (g) can be found at this age. An invagination of cytoplasm can be seen in one of the nuclei (arrow). Though the fixation here is improved over s-collidine buffered osmium, occasional breaks in the cell membranes can still be found. Fixed with glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 17,500. Fie. 6. A higher magnification of a junctional site similar to those seen in Fig. 5. The membranes are separated by approximately 200 A at these two sites (is). The cytoplasm adjacent to the junctional site appears increased in density. Clusters of free ribosomes (fr) and some membrane-bound ribosomes (er) are also seen in this micrograph. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 53,400.
~ !i~i¸~ ¸¸5¸i¸11
360
T. K. GREENLEE, JR. AND R. ROSS
w e r e seen b e n e a t h the e p i t h e l i u m of the l i m b b u d . T h e s e c o n s i s t e d of c a p i l l a r y e n d o t h e l i u m a n d w h a t a p p e a r e d to be i m m a t u r e m e s e n c h y m a l cells (Fig. 5). F o c a l j u n c t i o n a l sites, similar in a p p e a r a n c e to t h e i n t e r m e d i a t e j u n c t i o n s d e s c r i b e d in e p i t h e l i u m (4), w e r e o b s e r v e d b e t w e e n these m e s e n c h y m a l cells (25). I n t h e s e r e g i o n s the cell m e m b r a n e s
w e r e n e a r l y p a r a l l e l a n d s e p a r a t e d by a d i s t a n c e o f
a p p r o x i m a t e l y 200 •. B o t h the i n t e r v e n i n g space a n d the c y t o p l a s m a d j a c e n t to t h e j u n c t i o n a l sites w e r e i n c r e a s e d in d e n s i t y (Fig. 6). A t this e a r l y stage of d e v e l o p m e n t the n u c l e u s o c c u p i e d t h e m a j o r p a r t of e a c h of t h e s e cells. T h e c y t o p l a s m c o n t a i n e d m i t o c h o n d r i a , a few c i s t e r n a e of r o u g h e n d o p l a s m i c r e t i c u l u m a n d i s o l a t e d G o l g i vesicles. M o s t of the c y t o p l a s m h o w e v e r w a s o c c u p i e d b y n u m e r o u s clusters of free r i b o s o m e s (Figs. 5 a n d 6).
FIG. 7. This electron micrograph demonstrates tendon cells in a longitudinal section of a 16-day fetus. Collagen fibrils (co) can be identified in the extracellular space by their characteristic banding. A second population of unbanded fibrils (f) (100 ~ diameter) can also be seen. Rough endoplasmic reticulum (er) and Golgi complex (g) are more abundant at this stage than in the mesenchymal cells of the 12-day fetus (Fig. 5), but clusters of free ribosomes (fr) are still prominent. The cell nucleus (n) and mitochondria (m) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 18,200. FrG. 8. In an electron micrograph of a transversely sectioned 16-day fetal tendon, 100 /~ fibrils 0 c) of young elastic fibers are seen in the extracellular space. An obliquely sectioned junction site (is) between two of the cells can be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 16,000. FIG. 9. This low power electron micrograph of a transverse section from an 18-day fetal tendon demonstrates the area of junction between the tendon proper (Tendon) and the circumferentially oriented cells surrounding it. Fig. 10 is a light micrograph of a 1 t~ section taken adjacent to the thin section seen in Fig. 9. The circumferential cells closely applied to the tendon will become part of the visceral synovium (Vis. Syn.) and the cells at the top of the picture accompanied by longitudinally oriented collagen fibrils will become the fibrous sheath (Fib. Sheath). The sheath cavity will form in the area lying between these two regions. The cells in this region will segregate. Some of them will become the parietal synovium, and the others will remain as the visceral synovial layer of the tendon proper. No clear line of cleavage between these two can be seen at this stage. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 4500. FIG. 10. This light micrograph of a 1 # section taken after the thin section used for Fig. 9 shows the tendon surrounded by the circumferentially oriented cells (cc) that will form the synovial sheath and visceral synovial layer of the tendon (Ten.). Fixed in osmium tetroxide. Stained with azure II methylene blue. x 276. FIG. 11. There has been a marked change in the depth of the tendon in the 18-day fetus, as compared, to earlier stages, as seen in this electron micrograph of a transverse section. The increase in numbers and diameter of collagen fibrils (co) has caused the cells to separate and maintain contact at junctional sites found at the end of long cytoplasmic processes. Numerous collections of 100 A fibrils representing early elastic fiber (el) formation can also be seen. The rough endoplasmic reticulum and Golgi complex are more prominent in the tendon fibroblasts at this time. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 5700. FIG. 12. This micrograph demonstrates part of the tendon proper from and 18-day fetus similar to that seen in Fig. 11 demonstrating that in longitudinal section the cells are more spindle shaped than those seen in Fig. 7. The amount of collagen (co) is also increased. The contents of the cisternae of rough endoplasmic reticulum (er) are denser than the remainder of the cytoplasm, and free ribosomal clusters may still be observed. The nucleus (n) and mitochondria (m) can also be identified. Fixed in glutaraldehyde containing alcian blue. Stained with uranyl acetate and lead. x 13,500.
8
364
T . K . GREENLEE~ JR. AND R. ROSS
Sixteen-day fetus. The 16-day fetal tendon anlage did not contain a layer of circumferential cells as was found at the later stages. Collagen fibrils of approximately 200 A diameter were seen in the extracellular spaces between the cells of the tendon. A second population of fibrils approximately 100 ]t in diameter that did not display the characteristic banding of collagen was also present (Figs. 7 and 8). These fibrils were presumed to represent the precursors of elastic fibers subsequently identified in the older animals (11). The fibroblasts of the tendon contained many more profiles of rough endoplasmic reticulum and Golgi regions than did the less mature cells on the twelfth day of development. However, many free ribosomal clusters were still present within these cells and junctional sites were seen between adjoining fibroblasts (Fig. 8). Eighteen-day-fetus. After 18 days of fetal development the forming tendon was surrounded by a layer of circumferentially oriented cells 8-10 cells thick. At a later stage of development these cells will separate and be segregated into three separate structures. The innermost cells will remain with the tendon to become its visceral synovium. The outermost will become the fibrous sheath, and the cells remaining between will segregate at the time of cavity formation to produce the parietal synovium. No clear separation between these layers was apparent at this stage (Figs. 9 and 10). As in the tendon proper, there are junctional regions between these circumferentially oriented cells, but not between the sheath cells and the fibroblasts in the mass of the tendon. The amount of collagen between the cells by this time had increased, forcing the cells farther apart. The fibroblasts retain their intercellular contacts through junctional sites located at the ends of relatively slender cell processes (Figs. 11 and 13). At this stage the collagen fibrils were approximately 250 A in diameter and the second population of extracellular fibrils (100 A diameter) formed larger aggregates than those seen in the 16-day-old fetuses (Fig. 14). No qualitative changes were apparent in cell organelles, but there appeared to be a relative increase in the amount of rough-surfaced endoplasmic reticulum and a decrease in the number of clusters of free cytoplasmic ribosomes (Figs. 11 and 12). Newborn. As stated previously, the tendon sheath cavity was present at birth (Figs. 4 and 15). The visceral synovium consists of circumferentially oriented cells and collagen fibrils (Fig. 15). At this age, tendon cells appeared to be more compressed owing to the increase in extracellular fibrils. Cell-to-cell contact, however, was maintained by thin cytoplasmic processes attached by junctional sites similar to those previously described (Fig. 16). Concomitant with the increase in extracellular material, the fibroblasts appeared fusiform in longitudinal section in contrast to their appearance at the earlier stages of formation (Fig. 17). Two changes could be seen in the extracellular connective tissue elements at this
TENDON DEVELOPMENT
365
Eio. 13. This micrograph demonstrates a higher magnification of one of the intercellular attachment sites from an 18-day fetal tendon. The membranes are separated by approximately 200 ~, and the cytoplasm is denser adjacent to these sites. Fixed in glutaraldehyde containing alcian blue. Stained with uranyl acetate and lead. x 79,100. FI~. 14. In this micrograph from an 18-day fetus two transversely sectioned early elastic fibers (ela and elb) are shown. The 100 ~ fibrils (f) which constitute the major component of the immature elastic fiber are tubular in profile. Elastic fiber a (ela) is made up solely of 100 N fibrils while elastic fiber b (elb) contains a few small amorphous central areas (ca) surrounded by 100 N fibrils. Collagen fibils (co) of approximately 250 N diameter are present in the extracellular space. The collagen fibril (arrow) surrounded by a membrane is in the extracellular space. The relatively dark staining of the collagen fibrils and central areas of the elastic fibers seen here compared with those in Figs. 16 and 22 is characteristic of tissue subjected to glutaraldehyde fixation as compared with primary osmium fixation. Fixed in glutaraldehyde containing alcian blue, postfixed in osmium tetroxide. Stained with uranyl acetate and lead. x 89,500.
time: (a) The collagen fibrils were increased in diameter (430 A) a n d were relatively u n i f o r m in size. The 100 ~ fibrils that were observed from the sixteenth day of fetal life appeared to be part of a more complex structure at the time of birth. These structures consist of central a m o r p h o u s cores that stain poorly with u r a n y l acetate a n d / o r lead s u r r o u n d e d by the 100 ~ fibrils. The latter are t u b u l a r in cross section a n d are beaded in l o n g i t u d i n a l section (Figs. 16 a n d 17). The aggregates of these two c o m p o n e n t s can be identified as elastic fibers as a result of their correspondence 2 4 - - 6 7 1 8 2 3 J . Ultrastructure Y~esearch
366
T . K . GREENLEE, JR. AND R. ROSS
to s t r u c t u r e s w h i c h stain w i t h a l d e h y d e f u c h s i n in l i g h t m i c r o s c o p i c p r e p a r a t i o n s a n d t h e i r s i m i l a r i t y to elastic fibers seen in b o v i n e f e t a l l i g a m e n t u m n u c h a e (11). I t w a s f o u n d t h a t w h e n sections w e r e s t a i n e d w i t h u r a n y l acetate, M i l l o n i g ' s lead, o r p h o s p h o t u n g s t i c acid, c h a r a c t e r i s t i c s t a i n i n g r e a c t i o n s w e r e n o t e d f o r t h e t w o c o m p o n e n t s of the elastic fiber as well as f o r t h e c o l l a g e n fibrils. T h e 100 A fibrils s t a i n e d w i t h l e a d a n d / o r a q u e o u s u r a n y l a c e t a t e b u t n o t w i t h p h o s p h o t u n g s t i c acid, w h e r e a s t h e central core regions stained with phosphotungstic acid but not with lead or uranyl acetate. I n c o n t r a s t , t h e c o l l a g e n fibrils s t a i n e d w i t h u r a n y l a c e t a t e a n d p h o s p h o t u n g stic a c i d b u t n o t w i t h lead. Rough
e n d o p l a s m i c r e t i c u l u m a n d G o l g i vesicles w e r e m o r e p r o m i n e n t in t h e
t e n d o n f i b r o b l a s t s at this age t h a n in t h e 18 d a y fetuses, a n d free r i b o s o m a l a g g r e -
FIG. 15. This electron micrograph demonstrates the relationship between the tendon proper and the visceral synovium in the newborn animal as seen in transverse section. The collagen fibrils (cot) and the cells of the tendon proper (Ten. C) are transversely sectioned while the synovial cells (Syn. C) and their accompanying fine collagen fibrils (cos) are longitudinally oriented. The sheath cavity (Car.) is superficial to the synovial cells. Junction sites (ys) can be seen between the cells of the tendon, but not between the tendon cells and the synovial cells. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 12,400. Fla. 16. Two cells in this transverse section of newborn tendon still maintain contact by fine cytoplasmic processes. Junction sites (is) similar to those seen earlier are present. Both collagen fibrils (co) and elastic fibers (el) are found in the extracellular space. The rough endoplasmic reticulum (er) and Golgi Complex (g) are more prominent. Inset shows a higher magnification of one of the elastic fibers. This fiber consists of tubular fibrils (f) (100 A in diameter) and central cores (ca) that are almost amorphous in appearance. The elastic fiber in this micrograph is found in a cytoplasmic niche, a frequent finding in the tendon. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 21,400. Inset × 43,700. FIG. 17. When an elastic fiber from a newborn such as that seen in Fig. 16 is sectioned longitudinally, it appears as shown in this micrograph. The 100 A fibrils (f) do not contain apparent crossbanding as do the collagen fibrils (co) but they do appear to be beaded. The central core areas (ca) seen in cross section now appear as streaks. The elastic fiber shown here is adjacent to the cell membrane and runs a straighter course than do the collagen fibrils in the micrograph. The ergastoplasm (er) and Golgi complex (g) are prominent in the fibroblast. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 22,400. FIG. 18. This electron micrograph of a transverse section of a 30-day-old rat tendon demonstrates the marked increase in collagen (co). The cells are farther apart, but fine cytoplasmic processes (p) course between the bundles of fibrils and maintain cell contact. In some regions a mantle of fine filaments (fi) now separate the plasma membranes of the cells from the collagen. Abundant rough endoplasmic reticulum (er) and Golgi complex (g) are seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 15,000. FIG. 19. In this micrograph of a longitudinal section of a 30-day-old rat tendon, the cells tend to lie in rows separated by large bundles of collagen fibrils (co). Between adjacent cells the extracellular space is filled with fine filaments (fi). At this age the cytoplasm of the cells is packed with lamellae of rough endoplasmic reticulum (er) and Golgi complex (g). Mitochondria (m) are present, and a lipid droplet (l) can be seen in one of the cells. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 15,000. FIG. 20. This is a higher magnification of two adjacent cell membranes from a longitudinal section of a 30-day-old rat tendon. The numerous peripheral vesicles (v) were not a prominent feature in the earlier specimens. Fine filaments (fi) are prominent between the cells, and the rough endoplasmic reticulum (er) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. × 42,500.
TENDON DEVELOPMENT
367
!~!~~ii!iii,'i~i~ !!ii¸,~i~iii~t¸¸¸ ~ p
• ~ ~
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372
T . K . GREENLEE, JR. AND R. ROSS
gates, although diminished in number, were still present. Peripheral vesicles were occasionally seen in these cells as well. Thirty-day-old animals. At this stage the cellularity of the tendon was decreased in association with the increase in amount of extracellular collagen. Many fine cytoplasmic processes could be seen to course obliquely between the bundles of collagen. Presumably their function is to maintain cell contact (Fig. 18). The amount of rough endoplasmic reticulum and Golgi components had increased as compared with the newborn, and few, if any, free ribosomal aggregates were seen. Occasional lipid droplets and numerous peripheral vesicles were also present in these cells (Figs. 19, 20, and 22). A finding not previously observed was the presence of a mantle of fine extracellular filaments between adjacent tendon cells and between the plasma membranes of fibroblasts and the collagen fibrils (Figs. 12, 20, and 22). The collagen fibrils had continued to increase in size although the range of diameters (800-1600 A) was less homogeneous than that observed during the earlier stages of development (Fig. 22). In contrast, the elastic fibers had not changed in overall diameter (about 2500 ~ ) although the area of each fiber occupied by the central core regions had increased (Figs. 21 and 22). DISCUSSION The adult synovial sheath tendon contains three components that together constitute the functional tendon. These are: (a) the tendon proper, (b) the visceral synovium adherent to the tendon proper, and (c) the parietal synovium and sheath. Such a tendon is derived from a homogeneous population of cells that in the 12-day fetus appears to consist of relatively undifferentiated mesenchymal cells. During development, these cells differentiate into the three distinct populations that will form these three structures, all of which can be identified by birth.
Intercellular relationships Specific sites of cell attachment can be seen between the connective tissue cells within each of these three structures. Yet no sites of cell attachment were observed between the cells of any two neighboring structures, such as between the tendon proper and the visceral synovium, or between the cells of the visceral synovium and those in the parietal synovium. Thus, it can be concluded that the presence of contact sites plays a role in the maintenance of structural integrity within each of these components. As tendon motion begins, the parietal and visceral synovial cells are segregated, resulting in a sp.ace between the visceral and parietal synovia. A similar conclusion
TENDON DEVELOPMENT
373
FIG. 21. Elastic fibers are difficult to find in longitudinal sections of the 30-day-old rat tendon. This micrograph shows two obliquely sectioned fibers. The amorphous central areas (ca) are larger and more prominent at this age than in the newborn seen in Fig. 17. The 100/k fibrils (f) are still present and are unbanded as compared to the collagen (co). Note the cell process (p) adjacent to the elastic fibers. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 16,000. FIG. 22. If elastic fibers such as those seen in Fig. 21 are transversely sectioned, they appear as seen in this micrograph. The central core areas (ca) of these two fibers take up a larger percentage of the fiber than in Fig. 16, and the 100/~ fibrils (f) are less prominent. Elastic fibers remain in close proximity to the plasma membrane of the cells. Collagen fibrils (co) are less homogeneous in size than in the newborn and are greatly increased in diameter (800-1600/~). The cell nucleus (n), rough endoplasmic reticulum (er), and peripheral vesicles (v) can also be seen. Fixed in osmium tetroxide. Stained with uranyl acetate and lead. x 34,000.
was r e a c h e d b y Shields
(27) in a n e a r l y light m i c r o s c o p e s t u d y of d e v e l o p i n g pig
s y n o v i a l t e n d o n s . S y n o v i a l c a v i t y f o r m a t i o n a p p e a r s to o c c u r in a s i m i l a r f a s h i o n in t h e d e v e l o p m e n t of s y n o v i a l j o i n t s in c h i c k e m b r y o s
(14).
Forces acting upon the tendon during development It m a y be t h a t the o r i e n t a t i o n of t h e cells in e a c h of t h e c o m p o n e n t s of the t e n d o n is a f f e c t e d b y t h e f o r c e t h a t is a p p l i e d to it a n d is t r a n s m i t t e d i n t e r c e l l u l a r l y v i a the cell a t t a c h m e n t sites. T h e t e n d o n p r o p e r is a t t a c h e d to b o t h b o n e a n d m u s c l e . T h e r e -
374
T . K . GREENLEE, JR. AND R. ROSS
fore, the forces resulting from bone growth and muscle contraction would be transmitted from cell to cell in a longitudinal direction and would result in cells and extracellular fibrils oriented parallel to the developing bony rays of the foot. In contrast, the visceral synovial cells surround the tendon and contain no attachments to either the tendon proper, the bone, or the muscle. The forces acting upon this group of cells would result from the increase in girth of the tendon and would be approximately at a right angle to the longitudinal forces acting upon the tendon proper, resulting in a circumferentially oriented structure. The parietal synovium and sheath together form a horseshoe-shaped structure that is attached along the bones of the ray and surrounds the tendon. As with the visceral synovium, the primary force affecting the orientation of these cells will be the increasing girth of the tendon during development.
Cell differentiation During the period of tendon growth and maturation from the 12-day embryo to birth, several changes occur in the number and distribution of organelles within the fibroblasts of the tendon proper. These consist of (a) a decrease in the number of free aggregates or ribosomes; (b) an increase in the amount and distribution of rough endoplasmic reticulum (the cisternal membranes of the mature fibroblast characteristically contain large aggregates of attached ribosomes, often appearing as double rows or in a spiral formation), (c) an increase in the Golgi complex; and (d) an increase in the amount of peripheral vesicles. These changes are similar to those described by Goldberg and Green (9) in tissue culture fibroblasts during the transition from a proliferative phase of growth, when relatively little collagen was synthesized, to a stationary growth phase when collagen synthesis was actively taking place. The poorly differentiated mesenchymal cells in the 12-day embryo are similar in appearance to the early cells described by Hay (12, 13) and Salpeter (26) in the regenerating amphibian limb bud, as well as to poorly differentiated cells studied in other tissues (2, 15, 17, 19, 29). As in other investigations, it would appear that the development of an extensive rough endoplasmic reticulum can be correlated with the enhanced ability of these fibroblasts to synthesize and secrete the extracelhilar substances of the connective tissues of the tendon, i.e., collagen, elastin, and various protein polysaccharide complexes (5, 8, 20, 22, 24, 28). The role played by the numerous peripheral vesicles, particularly preminent near the plasma membrane of the fibroblasts in 30-day-old animals, is not clear. It is possible that they may be related to either pinocytosis or the secretion of extracellular materials; however, no evidence in this regard can be provided by this study.
TENDON DEVELOPMENT
375
The extracellular components Collagen fibrils. Collagen fibrils were present between the cells of the flexor tendons in 15- and 16-day fetuses. These fibrils were approximately 160 • in diameter. By 18 days of fetal life, the fibrils had increased in quantity and were between 200 and 250 A in diameter. A further increase in fibril diameter to approximately 400 A was observed at birth. At one month of age fibril diameters had become less homogeneous in the tendon and had increased markedly in size (800-1600 ,~ diameter). Similar age changes were demonstrated by Fitton Jackson (7) in her study of developing chick tendons. Elastic fibers. Of interest in this study was the presence of elastic fibers that reached a m a x i m u m diameter of approximately 2500 A. These fibers consisted of central a m o r p h o u s cores surrounded by 100 /~ tubular fibrils. The fine structure of these elastic fibers were described in detail in a previous publication (11). The function of these fibers within an inelastic structure such as a tendon is not clear. In the spinal ligaments and the connective tissues of arteries, elastic fibers predominate, whereas in tendon collagen fibrils are the major structural component. It is possible that the relatively small number of elastic fibers in tendon may play a role in the maintenance of a rest relationship between collagen fibrils following vigorous muscular contraction. Relatively little attention has been paid to this interesting aspect of tendon structure.
The authors would like to acknowledge the technical assistance of Dawn Bockus, Leslie Caldwell, Rene Collman, Ingrid Klock, and Franque Remington in the preparation of the thin sections; the photographic assistance of Johsel Namkung in preparation of the micrographs for publication; Sam Lewis for his help in providing the fetal rats; the secretarial services of Mrs. Dorris Knibb; and the editorial services of Miss Alison Ross. The authors are particularly indebted to Drs. Earl P. Benditt and John H. Luft for their help and encouragement during the progress of this study. REFERENCES
1. BENNETT,H. S. and LUFT, J. H., J. Biophys. Biochem. Cytol. 6, 113 (1959). 2. BEHNKE, O. and MOE, M., J. Cell Biol. 22, 633 (1964). 3. COMBS,J. W., Y. Cell Biol. 31, 563 (1966). 4. FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). 5. FEWER,D., THREADGOLD,J. and SHELDON,H., J. Ultrastruct. Res. 11, 166 (1964). 6. FERNANDO,N. V. and MOVAT, H. Z., Lab. Invest. 12, 214 (1964). 7. F~TTONJACKSON,S., Proe. Roy. Soc. B144, 556 (1956). 8. GODMAN,G. C. and LANE, M., J. Cell Biol. 21, 353 (1964). 9. GOLDBERG,B. and GREEN, H., J. Cell Biol. 22, 227 (1964). 10. GOMORI,G., Am. J. Clin. Pathol. 20, 665 (1950). 11. GREENLEE,T. K., JR., ROSS, R. and HARTMAN,J. H., J. CelIBiol. 30, 59 (1966).
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T.K. GREENLEE, JR. AND R. ROSS HAY, E., or. Biophys. Biochem. Cytol. 4, 583 (1958). Develop. Biol. 1, 555 (1959). -HENRIKSON,R. C. and COHEN, A. S., J. Ultrastruct. Res. 13, 129 (1965). HOWATSON,A. F. and HAM, A. W., Cancer Res. 15, 62 (1955). LUFT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). MARK, P. A., RIFKIND, R. A., and DANON, D., Proc. Natl. Acad. Sci. U.S. 50, 336 (1963). MmLON~G,G., J. Biophys. Biochem. Cytol. 11, 736 (1961). PALADE, G. E., J. Biophys. Biochem. Cytol. 1, 58 (1955). PALADE, G. E. and SmKEVITZ,P., J. Biophys. Biochem. Cytol. 2, 671 (1956). PEACH, R., WILLIAMS,G. and CHAPMAN,J. A., Am. J. Pathol. 38, 495 (1961). REVEL,J. P. and HAY, E., Z. Zellforsch. Mikroskop. Anat. 61, 110 (1963). RICHARDSON,K. C., JARRETT,L., and FINKE, E. H., Stain Technol. 35, 313 (1965). Ross, R. and BENDITT,E. P., J. Cell Biol. 27, 83 (1965). Ross, R. and GREENLEE,T. K., JR., Science, 153, 997 (1966). SALPETER,M. M. and SINGER, M., Proc. 5th Intern. Congr. Electron Microscopy, Philadelphia, 1962 Vol. 2, OO. Academic Press, New York, 1962. SHIELDS,R. T., Contrib. Embryol. 15, 53 (1923). SIEKEVITZ,P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 7, 619 (1960). SLAUTTERBACK,D. B. and FAWCETT, D. M.. J. Biophys. Biochem. Cytol. 5, 441 (1959). WASSERMAN,F., Am. J. Anat. 94, 399 (1954).