Calc. Tiss. P~es. 14, 15--29 (1974) 9 by Springer-Verlag 1974

An Ultrastructural Study of Bone Cells: The Occurrence of Microtubules, Microfilaments and Tight Junctions J e s s e M. W e i n g e r a n d M a r i j k e E. H o l t r o p Department of Orthopedic Surgery, Harvard Medical School, Children's Hospital Medical Center, Boston, Massachusetts Received March 3, accepted May 6, 1973

Bone cells of calvaria from young mice were studied at the ultrastructural level. Microtubules were demonstrated in both osteoblasts and osteocytes in the cell body, but not in the cell processes. Instead, the cytoplasm of cell processes is filled with bundles of 50 to 70 A microfilaments, running parallel to the long axis of the process. Where two cell processes meet, the cell membranes form a tight junction. These junctions are found between osteocytes, between osteocytes and osteoblasts, and between bodies of osteoblasts on the cell surface. The cell processes usually meet side-to-side, thus forming an extended tight junction. The junctions between osteoblasts are short and are believed to be spot-like. Inside the bone scarcely any extracellular space is visible. The likelihood of intracellular transport is discussed. Key words: Osteoeytes - - Osteoblasts - - Microtubules - - Microfilaments - - Intercellular Junctions. : Des cellules osseuses de calottes craniennes de jeunes souris sent 4tudi6es au microscope 61ectronique. Des microtubules sent visibles dans les ost6oblastes et les ost6ocytes dans le corps cellulaire, mais non dans les prolongements de la cellule. Le cytoplasme de ces prolongements est rempli de faisceaux de microfilaments de 50 s 70 A, parall~les b, l'axe longitudinal du prolongement. Au point de rencontre de 2 prolongements, les membranes cellulaires ferment une jonction 6troite. Ces jonctions s'observent entre les ost6ocytes, entre les ost6ocytes et les ost6oblastes et entre les corps des ost6oblastes, ~ la surface cellulaire. Les prolongements cellulaires habituellement se rencontrent cJte s cJte, formant ainsi une jonction 6troite 6tendue. Les jonctions entre ostJoblastes sent courtes et sent de type macula. A l'int6rieur de l'os, peu d'espace extra-eellulaire est visible. La probabilit6 de transport intracellulaire est envisag6e. Calvarien-Knochenzellen junger M~use wurden im ultrastrukturellen Bereich untersucht. Es wurden in den Osteoblasten und Osteocyten der ZellkJrper, nieht aber der Zellforts~tze, Mikrotubuli festgestellt. Dagegen ist das Cytoplasma der Zellfortsatze mit Bfindeln yon Mikrofasern yon 50--70 A geffillt, welche parallel der Liingsachse der Fortsatze angeordnet sind. An den Stellen, we zwei Forts~tze aufeinandertreffen, bilden die Zellmembrane eine feste Verbindung. Diese Verbindungen werden zwischen Osteocyten, zwisehen Osteocyten und Osteoblasten und zwischen OsteoblastenkJrper auf der Zelloberflache festgestellt. Die Zellforts/~tze verbinden sich im allgemeinen entlang ihren Seiten, wodurch eine lange feste Bindung entsteht. Die Verbindungen zwischen Osteoblasten sind kurz und vermutlich punktfJrmig. Innerhalb des Knochens ist kaum ein extracelluli~rer Raum sichtbar. Die Wahrscheinliehkeit des intracellul~ren Transportes wird diskutiert.

.For reprints: Marijke E. Holtrop, M.D., Ph.D., Department of Orthopedic Surgery, Children's Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115, USA.

16

J.M. Weinger and M. E. Holtrop

Introduction Bone n o t only serves a s u p p o r t i v e function, b u t also provides t h e calcium reservoir for m i n u t e - t o - m i n u t e a d j u s t m e n t of calcium level in blood a n d b o d y fluids. P a r a t h y r o i d hormone, which m e d i a t e s this process, increases t h e n u m b e r of osteoclasts, b u t this is d e m o n s t r a b l e o n l y after several d a y s a n d can, therefore, never a c c o u n t for t h e fast response resulting in calcium release from bone. Thus, o t h e r cells p r e s e n t a t a n y m o m e n t h a v e been t h o u g h t to be involved. I t has been p r o p o s e d t h a t osteocytes are d i r e c t l y involved in t h e process of calcium homeostasis (Talmage, 1969), a view t h a t has been s u p p o r t e d b y the observations of several i n v e s t i g a t o r s (Baylink a n d W e r g e d a l , 1971; B61anger, 1969). M e t a b o l i c a l l y active cells need a n efficient s y s t e m for t r a n s p o r t of n u t r i e n t s a n d waste products. W h e r e a s cells in tissues o t h e r t h a n bone are p r e s e n t in an intercellular g r o u n d substance w i t h a high w a t e r c o n t e n t t h a t m a k e s t h e m r e a d i l y accessible b y diffusion, t h e cells in bone are s u r r o u n d e d b y a calcified m a t r i x w i t h a v e r y low w a t e r content. There is l a c k of a g r e e m e n t b e t w e e n investigators concerning t h e a m o u n t of space p r e s e n t between cell a n d calcified m a t r i x , hence, t h e i m p o r t a n c e of this space for' diffusion is n o t clear. Osteocytes are connected w i t h each other a n d with t h e osteoblasts on t h e bone surface b y m e a n s of n u m e r o u s cell processes. There has been v e r y little irfformation on t h e u l t r a s t r u c t u r e of these processes. B61anger (1971) r e p o r t s o n l y t h a t t h e y "consist of a bundle of fine f i l a m e n t s " . According to Cameron (1971) " t h e y c o n t a i n filamentous m a t e r i a l a n d an occasional m i c r o t u b u l e " , a l t h o u g h t h e a c c o m p a n y i n g m i c r o g r a p h shows a m i c r o t u b u l e in t h e cell b o d y a n d n o t in t h e cell process. The r e c e n t finding of t i g h t j u n c t i o n s b e t w e e n cell processes (Holtrop a n d Weinger, 1971; W h i t s o n , 1972) opens t h e p o s s i b i l i t y of intercellular t r a n s p o r t a n d invites more a t t e n t i o n to structures w i t h i n t h e cell t h a t could p l a y a role in intra- a n d inter-cellular t r a n s p o r t . This p a p e r is a r e p o r t on t h e u l t r a s t r u c t u r e , occurrence a n d location of microtubules, filaments a n d t i g h t junctions in a n d b e t w e e n osteoeytes a n d osteoblasts a n d discusses t h e i r possible significance in bone cells. P a r t of this w o r k has been p r e s e n t e d elsewhere ( H o l t r o p a n d Weinger, 1971).

Materials and Methods The parietal bones of 23 Swiss albino mice, age 5 to 7 days, were used for this study. The bones were quickly dissected from anesthesized mice and immediately immersed in a mixture of one part 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 0 ~ and two parts of 1% OsO~ in 0.1 M cacodylate buffer, pH 7.4, at 0 ~ The bones were fixed for 3 h, washed in saline at 0~ and post-fixed for 3 h in 0.25% uranyl acetate in 0.1 M sodium acetate at 0~ brought to pH 6.3 with acetic acid. Following this the specimens were washed in saline, dehydrated in graded ethanols, infiltrated for one week in a mixture of epon and propylene oxide (1:1) and embedded in epon. Some specimens were fixed at room temperature for 2 h in 2.5% glutaraldehyde in 0.1 1~ cacodylate buffer, washed in cacodylate buffer, post-fixed at room temperature for 2 h in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4), washed again in cacodylate buffer and post-fixed in 0.25% uranyl acetate in 0.1 M sodium acetate ( p i t = 6 ) . Following this, the tissue was dehydrated and embedded as above. Thin sections were cut using diamond knives. Silver sections were picked up on parlodion-coated carbon reinforced copper grids and stained with a saturated solution of uranyl acetate in 50 % ethanol followed by 0.2 % lead citrate. The sections were viewed in a Siemens Elmiskop I electron microscope operating at 80 kV.

Fig. 1. M i c r o t u b u l e s

(rot)

a n d 50 to 70 A m i c r o f i l a m e n t s (arrows) in a n o s t e o c y t e fixed a t r o o m t e m p e r a t u r e , • 72 000

Fig. 2. L o n g i t u d i n a l sections of m i c r o t u b u l e s (rot) a n d cross sections of m i c r o t u b u l e s (arrows) in a n o s t e o c y t e fixed a t r o o m t e m p e r a t u r e , • 72000 2 Calc. Tiss. Res., Vol. 14

Figs. 3 and 4

Ultrastructure of Bone Cells

t9

Results Calvaria of young animals provide convenient material for an electron microscopic study for several reasons. The bone is thin, facilitating infiltration of fixatives and embedding materials, and thus ensuring tissue preservation. Also, calvaria are rich in bone cells compared to other young bones: in the thin layer of bone many osteocytes can be found. The outer su~ace of the bone is mainly covered with a layer of closely arranged osteoblasts, followed by layers of preosteoblasts and fibroblasts with numerous capillaries. The inner surface of the bone is mainly covered with undifferentiated lining cells and a few multinucleated osteoclasts. I n this study we have considered only the osteocytes and the layer of osteoblasts on the bone surface.

Cell Body In the cell body of both osteocytes and osteoblasts, microtubules and microfilaments can be recognized. The microtubules have a diameter of ~ 2 4 0 A and appear identical to the microtubules described in other cell types. They seem to run usually singly (Figs. 1, 2) and can be followed up to a length of ~2~z. Sometimes, small clusters of microtubules are seen running parallel to each other (Fig. 2). I t is not obvious, however, whether there exists any orientation of the microtubules relative to cell organelles or to cell polarity. The number of microtubules in any cell is difficult to assess, as they can be seen only in longitudinal or in cross-section and those cut tangentially are missed. As nothing is known about their orientation, the microtubules visible in a section are not a reliable percentage of the total number of microtubules in a cell. However, microtubules are seen in a sufficient number of sections to assume that they are present in every cell. I n some sections of osteoblasts we have seen a great many microtubules. Mierotubules were only seen after fixation at room temperature. With routine cold fixation, bundles of filaments with a diameter of 90 to 110 A were observed (Fig. 3), while these were only rarely seen after fixation at room temperature. I t is known that microtubules are labile structures and disintegrate at low temperatures (Tilney and Porter, 1967). Also, several investigators have reported that under a variety of conditions known to depo]ymerize microtubles, the loss of microtubules was correlated with an increase of 90 to 110 A filaments (Wisniewski et al., 1968; Ishikawa et al., 1968; Tilney et al., 1966). Thus, it seems likely that during the slow fixation of mineralized bone at 0 ~ the microtubules undergo change to form bundles of 90 to 110 A filaments. Microfilaments 50 to 70 A in diameter are present in both osteocytes and osteoblasts. They occur in small numbers and are randomly distributed, scattered through the cytoplasm (Fig. 1). In osteoblasts, bundles of parallel filaments can be seen along the cell membrane (Fig. 4). These filaments frequently extend

Fig. 3. 100 A filaments (/) in an osteoblast fixed at 0~ • 36000 Fig. 4. Detail of an osteoblast (ob) that shows microfilaments (arrows) along the cell membrane encircling a bundle of highly oriented collagen fibers in cross section, • 64000 2*

20

J . M . Weinger and M. E. Holtrop

Fig. 5. Cell process of an osteoblast filled with 50 to 70 A microfilaments (m[). An expanded area along the process contains several vesicles (v). The bone m a t r i x is partly calcified and collagen fibers (c]) are seen in cross section, • 66000 Fig. 6. Detail of an osteocyte cell process containing an abundance of microfilaments (m/). c/collagen fibrils, m m mineralized matrix, • 72000 Fig. 7. Cell process within mineralized matrix (ram) showing ribosomes (arrows) bound to endoplasmic reticulum membrane, • 90000

Ultrastructure of Bone Cells

21

into one or two cell processes, while the part of the cell containing the filaments seems to surround a bundle of extracellular collagen fibers oriented perpendicular to the plane of the filaments (Fig. 4). Two cells m a y take part in the "embrace ", the collagen fibers enclosed in this way being oriented more or less parallel and loosely fitted, or strictly parallel and close together (Fig. 4).

Cell Process Osteocytes have m a n y processes radially-directed around the cell and occupying canaliculi in the bone matrix. Osteoblasts on the bone surface also have processes, but only directed toward and extending into the bone. Bone cell processes can be long and branched. They are composed mainly of a bundle of microfilaments 50-70 A in diameter arranged parallel to the long axis of the cell process (Figs. 5, 6). At the junction of the process with the cell body, the filaments enter the cell body for a short distance, the bundle spreads and the filaments disappear from the plane of section (Fig. 6). The filaments occupy most of the space in the cell process. Sometimes, bulb-like swellings are seen in the process containing smooth vesicles of various sizes (Fig. 5). Occassionally, clusters of ribosomes can be seen, either free or bound to endoplasmic reticulum membranes (Fig. 7). Microtubules were not noticed in cell processes.

Relationship between Cells Osteocytes are connected with each other and also with osteoblasts on the bone surface. The contact is made by two processes meeting each other or by one process meeting a cell body. At the area of contact the membranes form a special structure which can be recognized as a cell junction (Figs. 8, 9, 10). The junctions are characterized by what appears to be a complete fusion of the outer leaflets of the apposing cell membranes, leaving no extracellular space (Figs. 8, 9). The area of fusion appears as a row of regularly spaced globules. The junctional membranes are more electron dense than nonjunctional membranes, and dense cytoplasmic material can be seen along the junction. Similar junctions are found between cell bodies of osteoblasts on the bone surface (Fig. 11). If the outer periosteal layer is stripped off the bone prior to fixation, the resolution of the cell membranes and the junction improve considerably. The leaflets of the unit membrane of the osteoblasts can now be clearly distinguished (Fig. 12), and the outer leaflets of the membranes at the junction do not appear as globules, but as a single intermediate line t h a t can be followed completely through the junction (Fig. 12). The width of all junctions is 135 to 200 A. The dense cytoplasmic fuzz along the junctional membrane makes accurate determination of the width difficult, hence a great variance in measurements has resulted. This type of junction, in either appearance, has been described and classified as a tight junction or zonula occludens (Brightman and Palay, 1963; Brightman and geese, 1969; Farquhar and Palade, 1963). The junctions between two cell processes in the bone usually have a considerable length due to the fact t h a t the processes meet side-to-side over some

22

J.M. Weinger and M. E. Holtrop

Figs. 8--10

Ultrastructure of Bone Cells

23

distance (Fig. 10). Moreover, the plane of the junction often seems not to be flat but undulating (Fig. 9, 10). I n this w a y a large area of surface contact between two cell processes is effected. Sometimes one can see an end-to-end or end-to-side contact (Fig. 8). Occasionally one cell process invaginates into part of a cell b o d y and in these instances, the complete circumference of m e m b r a n e contact forms a tight junction (Fig. 13). The junctions between osteoblasts are found where two cell bodies meet and are usually short (Fig. 11, 12). Because of the orientation of the junctional plane relative to the long axis of the cell process, the microfilaments run roughly parallel to the junctional plane (Fig. 9). I t could not be determined whether there is a n y a t t a c h m e n t of the filaments to the junction.

Relationship between Cell and Matrix The osteocyte lies in a lacuna and the osteocyte and osteoblast processes occupy canMiculi in the bone matrix. The osteocytes seem to fill the lacuna completely (Fig. 14, 15). Where space is found between cell and matrix, this seems to be due to poor fixation. The matrix adjacent to the lacuna is sometimes completely calcified, resulting in the complete encirclement of the osteocyte b y calcified bone (Fig. 15). Usually some osteoid is left between cell and calcified matrix ; the osteoid can be so extensive t h a t it surrounds the osteocyte completely (Fig. 14). I n the canaliculi the cell processes lie almost directly against calcified bone, and the extracellular space is minimal (Fig. 6, 9).

Discussion

The function of microtubules in cells has not yet been completely clarified. Microtubules have been related to two mechanisms: the development of cell shape (Byers and Porter, 1964; Tilney and Gibbons, 1969) and m o v e m e n t within the cell or cell transport (Freed and Lebowitz, 1970 ; L a c y et al., 1968 ; MacGregor and Stebbings, 1970; MMawista, 1965; Pelletier and Bornstein, 1972; Williams and Wolff, 1972). I n bone, where the cells lie within a rigid matrix, there is little chance for changing cell shape. I n these cells it is much more likely t h a t the microtubules are involved in transport. If this is so, it is surprising t h a t microtubules are not found in the cell processes.

Fig. 8. Tight junction between two bone cell processes (p). In cross section (arrows) the junction has 5 layers. At either side of the junction this structure is obscured by the tangential plane of the section, • 180000 Fig. 9. Side-to-side junction between two cell processes (/9). The plane of section intersects the undulating junctional plane twice, thus showing two cross sections of the junction (single and double arrows). The processes are composed predominantly of 50 to 70 A microfilaments that lie roughly parallel to the junction, • 92000 9 Fig. 10. An extensive side-to-side junction (arrows) between an osteoblast (ob) and an osteocyte process. The plane of section intersects the undulating junctional plane twice, compare with Fig. 9. (single and double arrows), • 35000

24

J . M . Weinger and M. E. Holtrop

:Fig. 11. Tight junction (arrows) between two osteoblasts (ob) on the bone surface. The fused outer leaflets of the membranes a t the junction appear as a row of globules, x 60000. Inset: Detail of the junction, x 180000 Fig. 12. Tight junction (arrows) between two osteoblasts (oh) on the bone surface, fixed after removal of the superficial layers of the periosteum. The leaflets of the uni~ membrane of the cells can be clearly distinguished and the fused outer leaflets of the membranes a t the junction appear as a continuous line instead of globules. I n the center the outer leaflets of the membranes are separated, thus interrupting the t i g h t junction, e8 extracellular space, x 100000. Inset: Higher magnification of the junction, X 240000

Ultrastructure of Bone Cells

25

Fig. 13. Invagination of a cell process (p) into an osteoblast (ob) on the bone surface. Along the complete circumference of membrane contact a tight junction can be seen, • 72000

I t has been demonstrated in many cell types that microfilaments with a diameter of 50 to 70 A are actin-like structures (Pollard et al., 1970 ; Bray, 1973) which are assumed to have contractile properties. I n osteoblasts a bundle of microfilaments is found adjacent and parallel to the cell membrane and extending into the cell processes. Their configuration and location resemble those of the filaments found in the contractile ring of dividing cells (Schroeder, 1970) which are considered actin-like because of their property of binding heavy meromyosin (Perry etal., 1971). The mierofilaments in osteoblasts are seen to encircle a bundle of collagen fibers and this relation suggests a mechanism of orientating the fibers : by contracting, the bundle would be squeezed and the fibers would be forced to lie parallel. In osteocytes microfilaments are found mainly in the cell processes where they exist in large numbers. I t is hard to conceive that these processes contract. I t has been proposed that contractile proteins, such as aetin, could provide the motive force for streaming in different systems, such as Nitella cells (Nagai and Rebhun, 1966), Physarum cells (Nachmias, 1973) and neuroblastoma cells (Burton and Kirkland, 1972). Until more data are obtained on the nature of the microfilaments in bone cells and the properties of contractile proteins in cells, one can only speculate on the function of mierofilaments in bone cell processes. The junctions found between bone cells correspond to the description of the tight junction or zonula occludens (Brightman and Palay, 1963; Farquhar and Palade, 1963). I n other cell types improved preservation of tight junctions was obtained after the use of uranyl acetate as a postfixative (Brightman and I~eese, 1969) and as a result a gap could be demonstrated in many tight junctions between the outer leaflets of the cell membranes (Brightman and Reese, 1969; l~evel and

26

J.M. Weinger and M. E. Holtrolo

Figs. 14 and 15

Ultrastructure of Bone Cells

27

Karnovsky, 1967). In bone cells uranyl acetate did improve the preservation of the tight junctions, but not effectively enough to show whether a gap was present. This was probably due to limited penetration of the uranyl acetate which could reach the layer of osteoblasts only after stripping the outer periosteal layer while cells within the bone were never reached. The total width of gap junctions is reported to be 135 to 150 A (Brightman and Reese, 1969). The total width of the junctions between bone cells is measured as 135 to 200 A, although it is likely that many junctions appear wider than they actually are because of dense cytoplasmic material along the junctional membrane. Thus the width of junctions between bone cells seem to be within the same range as that of gap junctions. The possibility of a gap in the junctions between bone cells is even more interesting in view of the fact that gap junctions are thought to be involved in intercellular communications (Payton et al., 1969; Revel and Karnovsky, 1967). There are indications that the junctions between bone cell processes might serve a communicating purpose; their location is such that large membrane areas come into close contact. Also, the plane of the junction is not flat, but often undulates (Figs. 9, 10), increasing the surface area even more. Hence, it seems likely that the tight junctions will prove to be gap junctions once better resolution can be obtained, and will appear to be involved in cell communication. In this respect, an interesting study was reported in which a dye injected into an osteoblast was seen to spread extremely rapidly into surrounding cells, including cells within the bone (Jeansonne et al., 1972), suggesting an active transport system between bone cells. Canas et al. have demonstrated that the ion concentration within bone differs from that outside bone and have proposed the idea of a bone fluid compartment controlled by a cellular barrier or membrane (Canas et al., 1969). The presence of tight junctions between ostcoblasts on the bone surface could provide such a cellular barrier, although this would only be true if the junctions formed a band all around the circumference of the cell. In that case the chance of finding a junction in any cross section would be great. However, the incidence of tight junctions between osteoblasts in sections in our material was relatively low and many cross sections of osteoblasts showed no junctions at all. This makes us suspect that the junctions are spot-like rather than band-like and cannot form a seal to a bony fluid compartment. Within the bone, the extracellular space around osteocytes and around cell processes seems to be minimal. I t has been demonstrated, however, that extracellular tracers such as horseradish peroxidase and pyroantimonate penetrate into the canaliculi and the osteocyte lacunae (Doty and Schofield, 1971). Thus, the possibility of extracellular diffusion within the matrix cannot be ruled out.

Fig. 14. Osteocyte in a lacuna that is surrounded by unmineralized matrix (c/ collagen fibers). The cell contains dilated cisternae (c) and mitochondria (m) with dense granules. A cell process (p) projects into a canaliculus, x 10800 Fig. 15. Osteocyte almost completely surrounded by mineralized matrix (ram), The cell contains a well developed Golgi area (G), dilated cisternae (c) and mitochondria @*) with dense granules, x 14400

28

J.M. Weinger and M. E. Holtrop

I n conclusion, microtubnles are found within bone cells t h a t could a c c o u n t for active flow of materials t h r o u g h the cells, while tight j u n c t i o n s between bone cells could provide passage from one cell to another. I n this way the osteoblasts on the cell surface a n d the osteocytes w i t h i n the bone would c o n s t i t u t e a n integrated cell system. The role of the mierofilaments in this system is n o t clear. Although the extracellular space is minimal, extraeellular diffusion c a n n o t be excluded.

Acknowledgments. This work was supported by the National Institutes of Health, Grant AM 15671 and The John A. Hartford Foundation. We wish to thank Ms. K. D. Altmann and Ms. E. L. Pickens for excellent technical assistance, and Mr. C. J. MacQuarrie for the photographic work.

References Baylink, D., Wergedal, J.: In: Cellular mechanism for calcium transfer and homeostasis, p. 257. New York: Academic Press 1971 B61anger, L.F.: Osteocytic osteolysis. CMcif. Tiss. Res. 4, 1-12 (1969) B61anger, L.F.: In: The biochemistry and physiology of bone, III osteocytic resorption, p. 240. New York~London: Academic Press 1971 Bray, D.: Cytoplasmic actin: A comparative study Cold Spr. Harb. Symp. quant. Biol. aT, 567 (1973) Brightman, M.W., Palay, S.L.: The fine structure of ependyma in the brain of the rat. J. Cell Biol. 19, 415-439 (1963) Brightman, M. W., l~eese, T. S. : Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 4@, 648-677 (1969) Burton, P. R., Kirkland, W.L.: Actin detected in mouse neuroblastoma cells by binding of heavy meromyosin. Nature (Lond.) New Biol. 239, 244-245 (1972) Byers, B., Porter, K. 1~. : Oriented microtubules in elongating cells of the developing lens rudiment after induction. Proc. nat. Aead. Sci. 52, 1091-1099 (1964) Cameron, D. A. : In: The Biochemistry and physiology of bone I, The ultrastructure of bone, p. 191. New York-London: Academic Press 1971 Canas, F., Terepka, A. P~., Neuman, W. F. : Potassium and milieu interieur of bone. Amer. J. Physiol. 27, 117-120 (1969) Dory, S. B., Schofield, B. H. : In: Calcium, parathyroid hormone and the calcitonins, p. 353. Amsterdam: Excerpta Medica 1971 Farquhar, M. G., Palade, G. E. : Junctional complexes in various epithelia. J. Cell Biol. 17, 375-412 (1963) Freed, J. J., Lebowitz, ~ . J. : The association of a class of sMtatory movements with microtubules in cultured cells. J. Cell Biol. 45, 334-353 (1970) Holtrop, M. E., Weinger, J.M.: Ultrastruetural evidence for a transport system in bone. In: Calcium, parathyroid hormone and the cMcitonins, p. 365. Amsterdam: Exeerpta Medica 1971 Ishikawa, H., Bischoff, P~., Holtzer, H. : Mitosis and intermediate-sized filaments in developing skeletal muscle. J. Cell Biol. 88, 538-555 {1968) Jeansonne, B.G., Feagin, F.F., Shoemaker, R.L., Rhem, W.S.: In: Fiftieth General Session, International Association for Dental Research, p. 173, 1972 (Abstract) Lacy, P. E., Howell, S. L., Young, D. A., Fink, C. J. : New hypothesis of insulin secretion. Nature (Lond.) 219, 1177-1179 (1968) MacGregor, H.C., Stebbings, It.: A massive system of microtubules associated with cytoplasmic movement in telotrophic ovarioles. J. Cell Sci. 6, 431-449 (1970) Malawista, S. E. : On the action of colchicine. J. exp. Med. 122, 361-384 (1965) Nachmias, V.T.: Physarum myosin: two new properties. Cold Spri. Harb. Symp. quant. Biol. 37, 567 (1973)

Ultrastructure of Bone Cells

29

Nagai, R., Rebhun, L. I. : Cytoplasmic mierofilaments in streaming Nitella cells. J. Ultrastruct. Res. 14, 571-589 (1966) Payton, B.W., Bennet, M. V. L., Pappas, G . D . : Permeability and structure of junctional membranes at an electrotonic synapse. Science 166, 1641-1643 (1969) Pelletier, G., Bornstein, M. D.: Effect of colchicine on rat anterior pituitary gand in tissue culture. Exp. Cell. ges. 70, 221-223 (1972) Perry, M.M., John, tI. A , Thomas, N. S. T. : Actin-like filaments in the cleavage furrow of newt egg. Exp. Cell Res. 65, 249-253 (1971) Pollard, T. D., Shelton, E., Welshing, R . R . , Korn, E. D.: Ultrastructural characterization of F-Actin isolated from Acanthamoeba castellanii and identification of cytoplasmic filaments as F-actin by reaction with rabbit heavy meromyosin. J. molec. Biol. 50, 91-98 (1970) Revel, J. P., Karnovsky, M. J. : Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 88, C7-C12 (1967) Schroeder, T. E. : The contractile ring. I. Fine structure of dividing mammalian (HeLa) cells and the effects of cytochalasin B. Z. Zellforsch. 109, 431449 (1970) Talmage, R. V.: The effects of parathyroid hormone on the movement of calcium between bone and fluid. Clin. Orthop. and Rel. I~es. 67, 210-223 (1969) Tilney, L. G., Gibbons, J. R. : Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. II. An experimental analysis of their role in development and maintenance of cell shape. J. Cell Biol. 41, 227-250 (1969) Tilney, L. G., Itiromoto, Y., Narsland, D. : Studies on the microtubules in heliozoa. III. A pressure analysis of the role of these structures in the formation and maintenance of the axopodia of Actinosphaerium nucleofilum (Barrett). J. Cell Biol. 29, 77-95 (1966) Tilney, L. G., Porter, K. R.: Studies on the microtubules in heliozoa. II. The effect of low temperature on these structures in the formation and maintenance of the axopodia. J. Cell Biol. 84, 327-343 (1967) Whitson, S. W. : Tight junction formation in the osteon. Clin. Orthop. and Rel. Res. 86, 206-213 (1972) Williams, J. A., Wolff, J. : Colchicine-binding protein and the secretion of thyroid hormone. J. Cell Biol. 54, 157-165 (1972) Wisniewski, H., Shelanski, M.L., Terry, R . D . : Effects of mitotic spindle inhibitors on neurotubules and neurofilaments in anterior horn cells. J. Cell Biol. 88, 224-229 (1968)