The U ltrastructure of Bone

Transcription

1 ANNALS O F CLIN IC A L AND LABORATORY SCIEN CE, Vol. 5, No. 4 Copyright 1975, Institute for Clinical Science The U ltrastructure of Bone MARIJKE E. HOLTROP, M D., Ph D. Department of Orthopedic Surgery, The Childrens Hospital Medical Center Boston, MA ABSTRACT The ultrastructure of the three specialized bone cells are described in relation to their function. Osteoblasts are the bone forming cells with an abundance of RER and a large Golgi area. Osteoblasts and osteocytes form an integrated cell system connected with each other by cell processes filled with actin-like filaments and joined by specialized cell junctions, probably gap junctions. Osteoclasts resorb bone. Characteristics are multiple nuclei, an abundance of mitochondria, a sparcity of RER and clusters of ribosomes. Lysosomes and vesicles contain hydrolytic enzymes. The resorbing area consists of a ruffled border completely encircled by a clear zone. Introduction In the metabolism of bone, two processes can be recognized that are related to the two funtions of bone tissue. First, the process whereby bone is constantly replaced in order to provide bone tissue with optimal supportive capacity. This is a slow and well integrated mechanism of breakdown of bone by osteoclasts followed by formation of bone by osteoblasts. The second metabolic process is related to the function of bone tissue in maintaining constant calcium levels in the body fluids. This involves a fast response to changing calcium needs in the organism. It is clear that this dual role of bone tissue requires a well organized structure with an integrated cell system. Three specialized cell types can be recognized: osteoblasts, osteocytes and osteoclasts. Osteoblasts and Osteocytes Osteoblasts are the cells involved in bone formation. They lie on the surface of the bone and show all the characteristics of a cell engaged in matrix formation (figure 1). An abundance of rough endoplasmic reticulum (RER) indicates the formation of proteins for secretion, i.e. mainly collagen. A well developed Golgi area is involved in the production of the ground substance of the matrix, the protein-polysaccharides. Microtubules can be found in the cytoplasm of osteoblasts,17 as well as in the cytoplasm of osteocytes17 and osteoclasts. The role of osteoblasts in the mineralization of the matrix has not been clarified yet. A layer of unmineralized matrix, the osteoid seam, can always be found between the osteoblasts and the mineralized bone (figure 1). In this osteoid seam vesicles have been described with an electrondense content, which are assumed to be derived from the osteoblasts and thought to form a nucleation site for the mineralization of the matrix.1 Some osteoblasts become encased in the newly formed matrix and are then called osteocytes (figure 2). This cell gradually decreases in size as a result of a decrease in cell organelles, mainly RER and Golgi. 264

2 U LTR A STR U CTU R E OF BONE 26 5 Figure 1. Osteoblasts on the bone surface. Characteristic is the abundance of rough endoplasmic reticulum (RER) and a large Golgi area (G). os: osteoid seam; mm: mineralized matrix, x 13,250.

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4 U LTR A STR U C TU R E OF BONE 267 Figure 3. Cell process filled with actin-like filaments, running through the osteoid seam (os) into a canaliculus in the mineralized matrix (mm), x 64,000. During this process matrix formation continues so that the osteocyte remains directly surrounded by matrix. Sometimes this matrix is completely mineralized, but usually some osteoid is still present. The mature osteocyte contains little RER, a small Golgi area and some undefined, membrane bounded electrondense bodies. The mitochondria sometimes contain elec trondense granules17 as are also found occa sionally in osteoblasts and osteoclasts and more frequently in chondrocytes of calci fying cartilage.413 The significance of these granules is not known but they have been implicated in calcium transport and/or cal cium storage.10 In addition to the formative stage of young osteocytes, a resorptive stage has been described in more mature os teocytes with changes in the cell structure and the surrounding matrix.6 The number of cells in the resorptive stage increased under influence of parathyroid hormone (PTH).7 These observations are consistent with the concept of osteocytic osteolysis whereby osteocytes are thought to play a significant role in calcium homeostasis.2 Osteocytes are connected with each other and with osteoblasts on the bone surface by numerous cell processes that run through canaliculi in the bone matrix. The cytoplasm of the processes is filled with microfilaments that run parallel to the long axis of the process (figure 3).17 These microfilaments have a diameter of 50 to 70 A and bind a heavy meromyosin9 which is a specific tech nique for the demonstration of actin-like filaments. Where two cell-processes meet, their cell membranes touch and form a spe cial structure which can be recognized as a specialized cell junction (figure 4).17 The processes usually meet side-to-side, thus forming an extended area of membrane

5 268 H O LTRO P Figure 4. Specialized membrane junction (arrows) between two cell processes in a canaliculus in the bone. The side-by-side contact provides an extended area of surface contact. The structure of the junction is partly obscured by the tangential plane of the section, x 180,000. contact (figure 4). As a result the microfila ments run roughly parallel to the junctions; it is not clear whether there is an attachment of the filaments to the junctional membrane or not. Owing to inadequate penetration of fixatives into the bone the type of junction cannot be classified with certainty, but it is most likely that these are gap junctions. The possibility of a gap in the junctions between bone cells is very interesting in view of the fact that gap junctions are thought to be in volved in intercellular communication, whereby molecules can move rather freely from one cell to another.1415 These junctions can also be found between the cell bodies of osteoblasts where they are short and are believed to be sport-like.17 In this way osteo blasts on the cell surface and osteocytes deep in the bone are all interconnected and form an integrated cell system. This would lend support to the concept of an active function of osteocytes in bone metabolism. Osteoclasts Osteoclasts have the function to resorb bone and as a result they differ greatly in ap pearance from osteoblasts. Osteoclasts are large multinucleated cells. Since sections for the electronmicroscope are so thin (ap proximately 600 A), the multiplicity of nu clei does not necessarily show up in the electronmicrograph. Other characteristics that are found throughout the cell and hence are seen in any section through the cell are the abundance of mitochondria, the sparcity of rough endoplasmic reticulum and

6 Figure 5. Detail of an osteoclast. Note the abundance of mitochondria (m) and the clusters of ribosomes (r). x 13,250.

7 270 H O LTRO P Figure 6. Resorbing area of an osteoclast with ruffled border (rb) and clear zone (cz). Note the vesicles (v) of variable size, x 9,000. the presence of many clusters of free ribosomes (figure 5). The resorbing area of the cell is formed by the ruffled border and a clear zone that completely encircles the ruffled border (figure 6).5 The ruffled border is considered to be the active site of resorption: the bone underneath the ruffled border is frayed and collagen fibres can be recognized by their characteristic binding. The ruffled border proper consists of deep invaginations of the cell membrane, which shows a coating that is not present on the membrane outside the ruffles.8 The clear zone is free of cell organelles and consists of fine granular material with bundles of fila ments running perpendicular to the bone surface and ending in small processes on the bone surface.9 These filaments bind to heavy meromyosin and hence can be considered actin-like.9 The bone underneath the clear zone is intact. The function of the clear zone may be to adhere the cell to the bone surface and form an isolated area for the ruffled border to excise its resorptive function. The resorption of bone is a complex process whereby calcium/phosphate, collagen fibres and the protein polysaccharides of the ground substance have to be broken down. The osteoclast contains granules and vesicles of variable number and size and often these are associated with the ruffled border.12,16 Several studies have indicated that lysosomes, vacuoles and infoldings of the ruffled border contain hydrolytic en zymes.3,11 It is likely that the bone is broken down in the ruffled border area outside the cell and that the products are invaginated and further degraded inside the cell. PTH enhances the resorptive activity of the osteoclast which reflects itself in an increase

8 ULTRASTRUCTURE OF BONE 271 of the ruffled border/clear zone area.5 Calcitonin reverses this process: the ruffled border decreases and the cell returns to its inactive state.5 References 1. ANDERSON, H. C. and REYNOLDS, J. J.: Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification. Develop. Biol. 34: , BELANGER, L. F. : Osteocytic osteolysis. Calcif. Tissue Res. 4:1-12, DOTY, S. B. and SCHOFIELD, B. H.: Electron microscopic localization of hydrolytic enzymes in osteoclasts. Histochem. J. 4: , HOLTROP, M. E. : The ultrastructure of the epiphyseal plate. II. The hypertrophic chondrocyte. Calcif. Tissue Res , HOLTROP, M. E., RAISZ, L. G., and SIMMONS, H. A.: The effects of parathyroid hormone, colchicine and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture. J. Cell. Biol. 60: , JANDE, S. S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks. J. Ultrastr. Res. 37: , JANDE, S. S.: Effects of parathormone on osteocytes and their surrounding bone matrix. Z. Zellforsch. 130: , KALLIO, D. M GARANT, P. R., and MINKIN, C.': Evidence of coated membranes in the ruffled border of the osteoclast. J. Ultrastr. Res. 37: , KING, G. J. and HOLTROP, M. E.: Demonstration of actin filaments in bone cells with heavy meromyosin. J. Dental Res. 53:245, LEHNINGER, A. L.: Mitochondria and calcium ion transport. Biochem. J. 119: , LUCHT, U.: Acid phosphatase of osteoclasts demonstrated by electron microscopic histochemistry. Histochemie 28: , LUCHT, U.: Cytoplasmic vacuoles and bodies of the osteoclast. An electron microscope study. Z. Zellforsch. 135: , MARTIN, J. H. and MATTHEWS, J. L.: Mitochondrial granules in chondrocytes. Calcif. Tissue Res , PAYTON, B. W., BENNET, M. V. L and PAPPAS, G. D.: Permeability and structure of junctional membranes at an electrotonic synapse. Science 166: , REVEL, J. P. and KARNOVSKY, M. J.: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell. Biol. 33.C7-C12, SCOTT, B. L.: The occurrence of specific cytoplasmic granules in the osteoclast. J. Ultrastr. Res. 19: , WEINGER, J. M. and HOLTROP, M. E.: An ultrastructural study of bone cells: the occurrence of microtubules, microfilaments and tight junctions. Calcif. Tissue Res. 14:15-29,1974.