Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1╬▒ 25-dihydroxyvitamin D3 have ruffled borders and resorb dentine.код для вставкиСкачать
THE ANATOMICAL RECORD 224379-391 (1989) Multinucleated Cells Formed on Calcified Dentine From Mouse Bone Marrow Cells Treated With 1a,25Dihydroxyvitamin D3 Have Ruffled Borders and Resorb Dentine TAKAHISA SASAKI, NAOYUKI TAKAHASHI, SHOHEI HIGASHI, AND TATSUO SUDA Department of Oral Anatomy (T.S., S.H.) and Biochemistry (N.T., T.S.), School Dentistry, Showa University, Tokyo 142, Japan of ABSTRACT Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of la,25-dihydroxyvitamin D3 [ la,25(OH),D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and a n extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited a n intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of k ~ ,2 5 (OH)~ D were 3 totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of la,25(OH),D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruff led borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1a,.%(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine. It is known that osteoclasts, the principal cells responsible for bone resorption, are multinucleated giant cells formed by fusion of mononuclear precursors derived from hematopoietic progenitor cells (Chambers, 1980; Bonucci, 1981; Roodman et al., 1985; Mundy and Roodman, 1987). The precise mechanism by which the progenitors in the hematopoietic cell population differentiate into osteoclasts, however, has not been established (Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et al., 1987; Takahashi et al., 1988a). Recently, we developed a mouse marrow culture system in which the progenitor cells fuse into multinucleated cells and have the following characteristics in common with authentic osteoclasts (Takahashi et al., 1988a). 1) The multinucleated cells formed exhibited tartrate-resistant acid phosphatase (TRACP) activity; TRACP is known as a marker enzyme of osteoclasts. 2) The formation of TRACP-positive multinucleated cells 0 1989 ALAN R. LISS, INC was markedly stimulated by osteotropic hormones such as la,25-dihydroxyvitamin D3 [ la,25(OH),D3] and parathyroid hormone. Calcitonin strikingly inhibited the formation of TRACP-positive multinucleated cells induced by both osteotropic hormones. 3) As in the case of authentic osteoclasts, TRACP-positive multinucleated cells were formed by fusion of mononuclear precursors. 4) Perhaps most important, when marrow mononuclear cells were cultured on calcified dentine slices in the Received June 7, 1988; accepted January 25, 1988. Address reprint requests to Dr. T. Sasaki, Department of Oral Anatomy, School of Dentistry, Showa University. 1-5-8Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Abbreviations used: la,25(OH)zD3, la,25-dihydroxyvitamin D,; TRACP, tartrate-resistant acid phosphatase; FCS, fetal calf serum; PBS, phosphate-buffered saline; a-MEM, alpha-minimal essential medium; rER, rough endoplasmic reticulum. 380 T. SASAKI ET AL. presence of l c ~ , 2 5 ( O H ) ~ the D ~ ,TRACP-positive multinucleated cells resorbed dentine by creating resorption lacunae. In the present study, we demonstrate further ultrastructural and cytochemical evidence that the multinucleated cells formed on dentine slices from mouse bone marrow mononuclear cells have ruff led borders and clear zones, the characteristic cytological features of functional osteoclasts, and they resorb calcified dentine in resorption lacunae exactly as do the authentic osteoclasts. MATERIALS AND METHODS Animals and Hormone Seven- to 9-week-old male mice and 3-day-old delete mice, both ddy strain, were obtained from Shizuoka Laboratories Animal Center (Shizuoka, Japan). l ~ i , 2 5 ( O H ) ~was D ~ the generous gift of Dr. I. Matsunaga, Chugai Pharmaceutical Co. (Tokyo, Japan). Bone Marrow Culture The mice were killed by cervical dislocation under light ether anesthesia, and their tibiae were aseptically removed and dissected free of adhering tissues. The bone ends were cut off with scissors, and the marrow cavity was flushed out with l ml of a-minimal essential medium (a-MEM) by slowly injecting a-MEM at one end of the bone using a sterile 25 gauge needle. The marrow cells were collected into tubes and washed twice with a-MEM. The marrow cells were then suspended in a-MEM containing 10% heat-inactivated fetal calf serum (FCS, Gibco, Grand Island, NY) at 1.5 x lo6 celldm1 and cultured for 8 days on either Lux coverslips (15 mm, Miles Scientific, Naperville, IL) or whale calcified dentine slices in 24-well plates (Sumitom0 Bakelite Co., Tokyo) (0.5 ml/well). The blocks of whale dentine were provided by Dr. A. Boyde, London University, London, UK. In brief, the dentine slices, 0.5-1 mm thick, were cut using a water-cooled diamond saw from blocks of sperm whale dentine, washed by ultrasonication for 10 min and further sterilized by ultraviolet light irradiation for 4 hr before use. Cultures were fed every 3 days by replacing 0.4 ml of old medium with fresh medium. 1a,25(OH)2D3(lop8 M) was added at the beginning of the culture and each time the medium was changed. All cultures were maintained at 37°C in a n atmosphere of 5% COz in air. Isolation of Osteoclasts and Alveolar Macrophages Osteoclasts were obtained from long bones of 3-dayold mice a s described previously (Takahashi et al., 1988b). Femora and tibiae of the mice were removed and curetted into a-MEM with 10% fetal bovine serum (eight bones/ml). The cell suspension was pipetted onto the center of Lux coverslips in 24-well plates (0.5 mll well) and incubated for 2 h r at 37°C. The coverslips were then washed with PBS and/or a-MEM and processed for electron microscopy. Alveolar macrophages were obtained by the tracheobronchial lavage method (Abe et al., 1983). Lavaged macrophages were washed with a-MEM, suspended in the same medium containing 5% heat-inactivated human serum a t a concentration of 5 x lo6 cells/ml, and plated in the center of the coverslips in 24-well plates (30 pUwe11). After incubation for 30 min at 37"C, nonadherent cells were removed, and only adherent cells were cultured in a-MEM with 5% human serum. M to la,25(OH)2D3was added to the cultures at induce fusion of alveolar macrophages. After culture for 4 days, the cells were rinsed with a phosphate-buffered saline (PBS, pH 7.4) and processed for electron microscopy. Electron Microscopy For conventional thin sectioning, adherent cells on either coverslips or dentine slices were rinsed with PBS and fixed with a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3% formaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) containing 0.05% CaC12. They were then rinsed in the same buffer overnight and postfixed with 1.5% potassium-ferrocyanide-reduced 1% osmium tetroxide for 2 h r at 4°C. Then, they were block-stained with 1% uranyl acetate in 10% ethanol for 30 min at room temperature, dehydrated through a graded ethanol series, and embedded as a monolayer in Poly Bed 812. For ultracytochemical detection of TRACP activity, we modified the medium of acid pnitrophenyl phosphatase (Miyayama et al., 1975). This medium consisted of 3 mM p-nitrophenyl phosphate (Sigma Chemical Company, St. Louis, MO) as a substrate, 3 mM lead nitrate as a capture reagent, 80 mM Tris maleate buffer (pH 6.7), 8% sucrose, and 10 mM sodium tartrate. Final pH was adjusted to 5.5-5.8 by adding 0.1 M nitric acid. Adherent cells on the coverslips were rinsed once with PBS and fixed in culture flasks containing a mixture of 1% glutaraldehyde and 1%formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h r at 4"C, rinsed in the same buffer overnight, and incubated for 30 min at room temperature (25°C) in the medium described above for TRACP staining. Control tissues were incubated in the same medium lacking substrate. After incubation, all specimens were postfixed with 1.5% potassium-ferrocyanide-reduced 1%osmium tetroxide for 10 min at 4°C and processed for embedding as described above. Sections perpendicular to the coverslips or the dentine slices were cut using a diamond knife on a Reichert-Jung Ultracut OmU-4 and counter-stained with aqueous uranyl acetate and lead citrate before being examined in a Hitachi HU-12A electron microscope at a n operating voltage of 75 kV. To study the inorganic elements in the above specimens, unstained ultrathin sections mounted on formvar-coated copper grids were analyzed with a Hitachi H-600 electron microscope fitted with a scanning device and Kevex 7000A energy-dispersive X-ray microanalyzer. Analysis points were selected using the scanning images of ultrathin sections. The microprobe conditions were 75 kV accelerating voltage, 2 x lop9A specimen current, 15 nm spot size, and 200 sec counting time. RESULTS Tartrate-ResistantAcid Phosphatase (TRACP) in lsolated Osteoclasts and Multinucleated Cells Derived From Either Alveolar Macrophages or Bone Marrow Cells TRACP activity was detected as electron-dense precipitations of lead phosphates in membrane-bounded OSTEOCLAST FORMATION I N MOUSE MARROW CULTURE bodies resembling lysosomes and cisterns of rough endoplasmic reticulum (rER) in isolated osteoclasts (Fig. la), but it was never seen in any subcellular structures of multinucleated cells derived from alveolar macrophages (Fig. lb). When marrow cells were cultured on coverslips in the presence of ~ c L , ~ ~ - ( Ofor H 8) days, ~ D ~a number of mature mononuclear cells and their fused multinucleated cells appeared (Fig. lc, d). The cytoplasm of the multinucleated cells and their mononuclear progenitors was characterized by the presence of many mitochondria, lysosomal membrane-bounded bodies, a moderate number of rER cisterns and stacks of Golgi membranes, numerous free polyribosomes, and several small membrane-bounded azurophilic granules. The cell surface formed numerous microvillous projections and complicated cytoplasmic membrane foldings associated with a number of pale vacuoles (Fig. lc-f). Unlike the multinucleated cells derived from alveolar macrophages, a n intense TRACP reaction was seen in the multinucleated cells and their precursors derived from bone marrow cells. In Figure lc, five mononuclear precursor cells undergoing fusion were recognized, and all of them exhibited TRACP activity. These structural and cytochemical features of mononuclear precursors were similar to those of multinucleated cells described below. In the multinucleated cells, TRACP activity was demonstrated in the rER cisterns (Fig. le) and membrane-bounded bodies resembling lysosomes (Fig. If) but was never found in the Golgi saccules and the plasma membranes. In the control experiments, omission of the substrate p-nitrophenyl phosphate from the incubation medium led to a complete disappearance of TRACP activity in all cell types, but omission of sodium tartrate did not alter the intensity and localization of TRACP in both isolated osteoclasts and marrow cell-derived multinucleated cells (data not shown). 38 1 with mononuclear progenitor cells (Fig. 2a, b). The mononuclear progenitor cells showed no membrane specialization for cell-to-coverslip contact. Multinucleated cells formed on coverslips also showed smooth cell surfaces devoid of any membrane specializations for cell-to-coverslip contact. The free cell surface facing the culture medium extended numerous long microvilli associated with a number of membrane infoldings and pale vacuoles of various sizes (Fig. 2c). Although these multinucleated cells contained many cell organelles, they appeared to lack the morphological polarity of cytoplasmic organization: the central part of cytoplasm of the multinucleated cells was occupied by many stacks of Golgi saccules (Fig. 2c, inset), mitochondria, and cisterns of rER, whereas nuclei having both euchromatin and heterochromatin were arranged along the peripheral cytoplasm. Nucleoli were clearly distinguished in these nuclei. Nuclear mitosis never appeared during the formation of multinucleated cells. Mitochondria, rER, free polyribosomes, and lysosomes were scattered throughout the cytoplasm of the multinucleated cells (Fig. 2c). The basal cell surface of the multinucleated cells facing the coverslip and its subsurface cytoplasm showed no membrane specializations, except for shallow plasmalemma1 infoldings (Fig. 2d, inset). Thus, it is concluded that the multinucleated cells formed on coverslips lacked ruffled borders. The cell periphery of the multinucleated cells showed a clear zone-like region devoid of most cell organelles, except for numerous free ribosomes and cytoplasmic filaments (Fig. 2d). Multinucleated Cells Formed on Dentine Slices When marrow mononuclear cells were cultured on dentine slices in the presence of l ~ i , 2 5 ( 0 H ) ~multiD~, nucleated cells with somewhat different morphological features were formed. These multinucleated cells were located on the shallow resorption lacunae of the dentine surface (Fig. 3a). In contrast with the multinucleMultinucleated Cells Formed on Coverslips ated cells formed on coverslips, those formed on dentine When bone marrow cells were cultured for 8 days with slices in vitro appeared to satisfy almost all the morlop8 M 1a,25(OH)2D3on coverslips, typical multinu- phological criteria of fully differentiated osteoclasts. The multinucleated cells had several nuclei with eucleated cells and their mononuclear progenitors were formed. On the coverslips, a number of mature mono- chromatin and heterochromatin and prominent nuclenuclear cells thought to be progenitors of multinucle- oli located at the cell periphery facing the culture meated cells were observed (Fig. 2a). The nucleus of such dium (Fig. 3a). Many stacks of the Golgi membranes, cells possessed both euchromatin and heterochromatin each stack consisting of about five Golgi saccules and and sometimes had a prominent nucleolus. The cyto- related vesicles, were localized in the perinuclear cytoplasm was characterized by the presence of many mi- plasm (Fig. 3b). The multinucleated cells exhibited tochondria, lysosomal membrane-bounded bodies, a well-developed ruff led borders consisting of deeply inmoderate number of rER cisterns, the stacks of Golgi folded plasma membranes adjacent to the dentine sursaccules, numerous free polyribosomes, and several face. The depth of these membrane infoldings was small membrane-bounded azurophilic granules. Facing 1.5-4 Km; the adjacent cytoplasm contained many pale the culture medium, the cells extended numerous long endocytotic vacuoles of various sizes and configuramicrovilli (pseudopodia). Another cell surface special- tions, some of which were apparently continuous with ization feature was the complicated deep membrane the narrow extracellular spaces of the ruffled border infoldings (Fig. 2a). These structural features of mono- (10-40 nm in width) (Fig. 3b, c). The extracellular space of the ruff led border was parnuclear progenitors were similar to those of multinucleated cells described below. The mononuclear cells tially filled with numerous small apatite crystals of appeared to fuse with each other (indicated by arrow dentine matrix. Many pale endocytotic vacuoles adjaheads in Fig. 2b). When these mononuclear cells came cent to or continuous with the ruffled border also coninto partial contact with the coverslips, osteoblast-like tained apatite crystals (Fig. 3b, c). Characteristically, cells appeared to intervene between the mononuclear the surface dentine layer facing the ruffled border excells and the coverslips and were in very close contact hibited lower crystal density compared with the deeper 382 T. SASAKI ET AL. Fig. 1. Ultracytochemical localization of tartrate-resistant acid phosphatase (TRACP). a: An isolated osteoclast exhibits intense TRACP activity in lysosomes and tubulovesicular structures. x 10,000. b A multinucleated cell formed from mouse alveolar macrophages in the presence of 1 1 ~ , 2 5 ( 0 H ) ~ItDis~totally . negative for the enzymatic reaction. x 6,250. c: Mononuclear bone marrow cells cultured with 1 1 ~ , 2 5 ( 0 H ) ~These D ~ . cells undergoing multinucleation show intense TRACP activity. Two mononuclear monocyte-like cells (arrowheads) are negative for the enzymatic reaction. x 3,750. OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE Fig. 1, d A multinucleated cell formed from bone marrow cells in the presence of l c ~ , 2 5 ( O H ) ~exhibits D~ TRACP activity. Note the presence of TRACP-positive mononuclear cells (arrowheads) around the 383 multi-nucleated cell. cs: coverslip. x 5,000. e: TRACP activity in cisterns of endoplasmic reticulum of a multinucleated cell. x 33,750. fi TRACP activity in lysosomes. x 27,000. 384 T. SASAKI ET AL. layer (Figs. 3b, 4a). The peritubular dentine had higher crystal density than the intertubular dentine. At the border between the electron-lucent surface dentine layer and the electron-opaque deeper layer, an electron-dense line of crystals appeared (Fig. 4a, arrows). Toward the peripheral region of the multinucleated cells facing the dentine surface, the so-called clear zone appeared adjacent to the ruffled border (Fig. 4b). The plasma membrane of the clear zone showed small undulations but never formed deep membrane infoldings. The cytoplasm of the clear zone was 0.5-1.2 pm in depth and totally devoid of cellular organelles, except for numerous cytoplasmic filaments and a small number of free ribosomes (Fig. 4b). The dentine surface fac- ing the peripheral clear zone contained more mineralized matrix similar to the deeper zone of dentine than that facing the ruffled border (Fig. 4a and b). X-ray microanalysis of dentine in various regions was also performed on the same but unstained sections. Several major elemental peaks of calcium (Ca: Ka = 3.691 KeV; KP = 4.012 KeV), phosphorus (P: Ka = 2.013 KeV), and sulfur (S: Ka = 2.307 KeV), as well as some minor peaks derived from plastic embedding medium (Cl), fixative (K and Os), and block-staining solution (Ur) were recognized. The lowest spectrum peaks of Ca, P, and S were obtained from the spaces of the ruffled border containing apatite crystals (Fig. 5a). The surface dentine layer showed moderate peaks of OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE 385 Fig. 2. a: A mononuclear progenitor cell has come in contact with a coverslip (CS) (marked by arrow). An osteoblast-like cell (OBC) intervenes between a mononuclear cell and a coverslip and has close contact with the mononuclear cell (arrowheads). The inset figure indicates the perinuclear cytoplasm of the mononuclear cell including the Golgi saccules (Go), mitochondria, azurophilic granules, and pale vacuoles. x 7,500; inset, X 25,000. b: Fusion of two mononuclear progenitor cells at marked areas (arrowheads). These cells have partial contract (arrows) with a coverslip (CS). An osteoblast-like cell (OBC) is present between the progenitors cell and a coverslip. x 10,000. c: A low-magnification view of a multinucleated cell formed on a coverslip (CS). The cell contains several nuclei and abundant cell organelles such as mitochondria and cisterns of rER. Many vacuoles are scattered throughout the cytoplasm. The cell surface facing the culture medium has a number of microvilli, while that facing a coverslip is quite smooth. The inset figure indicates the perinuclear cytoplasm of the multinucleated cell, including the stacks of Golgi saccules (Go) and pale vacuoles. ~3,750; inset, ~ 8 , 0 0 0 .d The peripheral cytoplasm of multinucleated cell facing a coverslip (CS) is devoid of cell organelles except for numerous free ribosomes and microfilaments. The cell surface at the central part of a multinucleated cell facing a coverslip (CS) shows shallow membrane infoldings (MI) but not ruffled border (inset figure). x 12,000. Ca, P, and S (Fig. 5b). The highest spectrum peaks of Ca, P, and S were detected from regions deeper in the dentine layer (Fig. 5c). The multinucleated cells contained various types of phagosomes and lysosomes. As has been described, many pale endocytotic vacuoles in the ruffled border zone contained apatite crystals (Figs. 3c, 6a). These vacuoles are regarded as phagosomes. Round dark lysosomes in the perinuclear cytoplasm also contained a smaller number of apatite crystals (Fig. 6b). Multivesicular bodies and coated vesicles were always free of apatite crystals. X-ray microanalysis of the pale en- 386 T.SASAKI ET AL. OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE docytotic vacuoles and dark lysosomes containing crystals detected energy spectrum peaks of Ca, P, and S to some degree, but the peak heights were apparently much lower than those in the surface and deeper dentine layers (inset figures in Fig. 6 a, b). The sulfur peak was sometimes not distinct in the dark lysosomes, while it was evident in the pale endocytotic vacuoles adjacent to the ruffled border (inset figures in Fig. 6a, b). 387 The bone resorption-stimulating hormone, 101,25 (OHhD3, is directly or indirectly involved in the differentiation and fusion of mononuclear precursors to form multinucleated cells (Abe et al., 1983; Bar-Shavit et al., 1983; Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et al., 1987). Recently, we developed a mouse marrow culture system, in which multinucleated cells with several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP), were formed (Takahashi et al., 1988a). The formation of multinucleated cells by 101,25(OH)~D~ was greatly inhibited by simultaneously adding salmon calcitonin. When marrow cells were plated on coverslip to start a culture, no multinucleated TRACP-positive osteoclastlike cells were found (Takahashi et al., 1988a). The multinucleated cells formed on coverslips in the presence of 101,25 (OH)ZD3 exhibited several ultrastructural features of osteoclasts such as abundant pleomorphic mitochondria, many stacks of Golgi saccules, an extensive system of microvilli and related membrane infoldings, and a peripheral cytoplasm lacking organelles (a clear zone-like structure) (Kallio et al., 1971; Gothlin and Ericsson, 1976; Holtrop and King, 1977; Ibbotson et al., 1984; MacDonald et al., 1987). As seen in isolated authentic osteoclasts, TRACP, a reliable marker enzyme of osteoclasts (Burger et al., 1982; Minkin, 1982; van de Wijngaert and Burger, 1986), was detected in the bone marrowderived multinucleated cells and their mononuclear precursors a t an ultrastructural level, using p-nitrophenyl phosphate as a substrate. Multinucleated cells formed from mouse alveolar macrophages in the presence of 101,25(OH)~D~ showed no TRACP activity. Acid trimetaphosphatase and f3-glycerophosphatase activities, reliable marker enzymes of lysosomes, were observed both in the alveolar macrophage-derived and in the bone marrow-derived multinucleated cells (data not shown). Previous cytochemical investigations of lysosomal acid phosphatases in osteoclasts have demonstrated the intense enzymatic activities of P-glycerophosphatase, arylsulfatase, and trimetaphosphatase in the Golgi-GERL-lysosomesystem of the osteoclasts and their precursors (Lucht, 1971; Doty and Schofield, 1972; Baron et al., 19861, but none of these acid phosphatase isoenzymes are specific for osteoclasts. The ultracytochemical demonstration of TRACP shown in this study is therefore thought to be a useful biological tool for determining whether they are osteoclasts and their lineage cells. It should be noted that the ruffled border, the most important morphological feature of functional osteoclasts, was not observed in the multinucleated cells formed on coverslips. When mononuclear marrow cells were cultured on dentine slices, the ruffled border was observed in the multinucleated cells that formed. We used dentine slices instead of bone slices, since dentine slices do not have any lacunae for osteocytes in the matrices, and there is no risk of misjudging them to be newly formed resorption lacunae (Boyde et al., 1984). Since ruffled borders were detected on the cell surface of the multinucleated cells facing dentine slices, it is suggested that some unidentified components of calcified dentine are responsible for inducing the formation of ruffled borders. The appearance of ruffled borders on multinucleated cells affects the cytoplasmic polarization. From the surface toward the inside of the cells, the functional arrangement, in order, is the ruffled border, an adjacent endocytotic vacuole region, a perinuclear Golgi region, and dark lysosomes in the deeper cytoplasm. This is the characteristic organization of the Golgi-lysosome system in osteoclasts (Kallio et al., 1971; Gothlin and Ericsson, 1976; Holtrop and King, 1977). We call these multinucleated cells fully differentiated functional osteoclasts. It is therefore likely that there are two morphological states of osteoclasts: 1)the common, non-functional osteoclast morphology and 2) the acquired specialized features characterizing functional osteoclasts. To induce the latter, calcified tissues such as bone and dentine are thought to be required. It is quite understandable that previous investigators have failed to demonstrate multinucleated cells with ruff led borders, since they cultured mononuclear marrow cells on plastic dishes or coverslips (Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et al., 1987). Our recent autoradiographic study using ['2511-labeled calcitonin has revealed that mouse marrow cell-derived TRACP-positive multinucleated cells and mononuclear cells grown on coverslips have calcitonin receptors as do authentic osteoclasts (Warshawsky et al., 1980; Takahashi et al., 198813). Therefore all morphological characteristics of osteoclast-like multinucleated cells, except the ruff led border, should be regarded as being non-functional basic features of osteoclasts. In the absence of calcified tissues in the culture, the marrow cell-derived multi- Fig. 3. a: A low-magnification view of multinucleated cell on a calcified dentine slice (CDe). This multinucleated cell has numerous mitochondria, cisterns of rough endoplasmic reticulum, many pale endocytotic vacuoles, and dark lysosomes. Facing the calcified dentine, the cell shows a characteristic ruffled border. x 3,750. b The perinuclear cytoplasm and the ruffled border zone (RB) of the same multinucleated cell as in a in a resorption lacuna on a calcified dentine slice (CDe). The cytoplasm contains several stacks of Golgi suc- cules (Go),several mitochondria (Mt), multivesicular bodies (MVB), and pale endocytotic vacuoles (V). The ruffled border is composed of a deeply infolded plasma membrane. The calcified dentine facing the ruffled border is reduced in crystal density. ~ 1 2 , 5 0 0 c: . A higher magnification of the ruffled border. Note that numerous small apatite crystals are present in the narrow extracellular space of the ruffled border and in adjacent newly formed pale endocytotic vacuoles (arrowheads). x 27,500. DISCUSSION 388 T. SASAKI ET AL. OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE 389 nucleated cells exhibited neither ruffled borders nor polarized organization of cytoplasmic organelles (Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et al., 1987). The components in calcified tissues that are responsible for inducing the ruffled border need to be determined. The present study also points out the resorption process of calcified tissues by multinucleated cells: liberation of crystals from the collagen fibers by decalcification of dentine and cellular absorption and intracellular degradation of liberated crystals. First, the surface dentine layer facing the ruffled border of the multinucleated cells is partially decalcified, resulting in numerous liberated apatite crystals. The surface dentine facing the clear zone is not decalcified and remains intact. X-ray microanalysis of the dentine overlaid by the ruffled border revealed that the peaks of Ca and P in the surface layer were lower than those in the deeper layer. The identical analytical results were obtained by in vivo dentine resorption mediated by human odontoclasts (Sasaki et al., 1988a,b). In addition, the structural porosity of the bone matrix facing the osteoclast ruff led border has been indicated by the penetration of intravenously injected horseradish peroxidase into the matrix zone from 1 to 2 pm below the surface (Sasaki et al., 1985). A partial decalcification of the bone surface under the osteoclast ruffled border has been recognized by the presence of loose apatite crystals and disrupted collagen fibers at that matrix zone; this decalcification is considered to be the first extracellular phase in bone resorption (Kallio et al., 1971; Bonucci, 1974). Such a partial decalcification of the bone matrix may result in liberation and release of apatite crystals from the matrix for subsequent resorption by osteoclasts. These findings agree well with a recent hypothesis that osteoclasts produce protons and hydrolytic enzymes, including acid protease, and secrete them into the local milieu between the ruffled borders and resorbing bone surface, to decalcify and degrade bone matrix (Fallon, 1984; Baron et al., 1986; Blair et al., 1986; Chambers et al., 1987). In fact, Fallon (1984) reported that the local pH in resorption lacunae on bone surfaces covered with ruffled borders of osteoclasts is below 5. Baron et al. (1985) and Silver (1988) have recently achieved similar results. The liberated apatite crystals of dentine are distributed in the extracellular canals of the ruffled borders, in the newly formed pale endocytotic vacuoles, and in the dark lysosomes of the multinucleated cells. The ruffled border of the multinucleated cells therefore appears to be the site of formation of endocytotic vacuoles containing apatite crystals. Absorption of apatite crystals by multinucleated cells is thought to be due to a fluid-phase endocytosis that has been demonstrated in the osteoclast ruffled border (Sasaki et al., 1985). Similar absorption of apatite crystals into the extracellular channels of the ruffled border has been observed in osteoclastic bone resorption (Bonucci, 1974; Gothlin and Ericsson, 1976). An X-ray microanalysis revealed that the apatite crystals showed lower peak heights of Ca, P, and S in the dark lysosomes than in the newly formed pale endocytotic vacuoles and the ruff led borders of multinucleated cells. In particular, the S peak was scarcely detected in the dark lysosomes, suggesting that digestion of the sulfur-containing material had taken place a t this phase. Therefore, the multinucleated cells are thought to resorb liberated apatite crystals with sulfated organic materials via the ruff led borders and endocytotic vacuoles and subsequently to digest them in lysosomes. It has also been well documented that the osteoclasts contain various lysosomal enzymes in membrane-bounded bodies around the Golgi complexes such as the dark lysosomes shown in this study (Lucht, 1971; Doty and Schofield, 1972, 1976; Walker, 1972; Rath et al., 1981; Baron et al., 1985, 1986). Takagi et al. (1982) have demonstrated that the complex carbohydrates of bone matrix are incorporated by osteoclasts into pale endocytotic vacuoles by way of the ruffled borders. These vacuoles then fuse with primary lysosomes derived from the Golgi vesicles and consequently become the phagolysosomes where intracellular digestion of the incorporated materials takes place. It is therefore likely that the pale endocytotic vacuoles containing apatite crystals in the multinucleated cells have obtained hydrolytic enzymes from the Golgi apparatus, resulting in the dark lysosomes, where incorporated materials are digested. Taken together with these results, osteoclasts are thought to resorb free inorganic crystals and dissolve them and to digest the organic matrix of bone in an acidic microenvironment mediated by the ruff led border. In conclusion, we developed a culture system to make functionally fully differentiated osteoclasts. This culture system provides a useful experimental model for further examining the mechanisms of osteoclast formation and bone resorption in normal and pathological states. Fig. 4. a: The surface dentine layer (marked by a bracket) facing the ruff led border (RB) consists of relatively electron-lucent intertubular dentine (ID) showing low crystal density and electron-dense peritubular dentine (I'D) surrounding the dentinal tubules (DT). Beyond the electron-dense border (arrows), both the intertubular and peritubular dentine matrices in the deeper layer show a much higher packing density of apatite crystals, x 15,000. b The surface dentine layer facing the clear zone (CZ) of multinucleated cell. Toward the peripheral region of the multinucleated cell (at the top right-hand corner), the surface dentine consists of densely packed crystals, whereas the crystal density becomes reduced in the dentine adjacent to the ruffled border region (RB, at the bottom left-hand corner). DT: dentinal tubule, x 12,500. Fig. 5. Energy spectrum peaks from an X-ray microanalysis of a: an area in the ruffled border, b the surface dentine layer facing the ruffled border, and c: the deeper dentine layer beyond the dense border in Figure 4a. The peaks of calcium (Ca) and inorganic phosphorus (PI derived presumably from apatite crystals were lowest in a: the ruffled border, highest in c: the deeper dentine layer, and intermediate in b: the surface dentine layer. The phosphorus peak is combined with the osmium peak ( 0 s ) . 390 T. SASAKI ET AL. Fig. 6. a: Four pale endocytotic vacuoles (arrowheads) adjacent to the ruffled border of a multinucleated cell containing apatite crystals. The inset figure indicates the high peaks of Ca and P and a low peak of sulfur (S) in these vacuoles. x 37,500. b Five dark lysosomes (arrowheads) containing crystal-like dense materials in the deeper cyto- plasm of a multinucleated cell. An X-ray microanalysis of this dense material revealed lower but significant peaks of Ca and P. The peak of S was almost undetectable. x 37,500. Os, osmium; Ur, uranyl acetate. OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE ACKNOWLEDGMENTS This study was supported by Grants-in-Aid (60440086, 61570894, and 61870047) from the Ministry of Science, Education, and Culture of Japan. LITERATURE CITED Abe, E., C. Miyaura, H. Tanaka, Y. Shiina, T. Kuribayashi, S. Suda, Y. Nishii, H.F. DeLuca, and T. Suda 1983 la,25-Dihydroxyvitamin D3 promotes fusion of alveolar macrophages both by a direct mechanism and by a spleen cell-mediated indirect mechanism. Proc. Natl. Acad. Sci. U.S.A., 805583-5587. Baron, R., L. Neff, D. Loubard, and P.J. Courtoy 1985 Cell mediated extracellular acidification and bone resorption: Evidence for a low pH in resorbing lacunae and localization of a 100 KD lysosoma1 membrane protein at the osteoclast ruffled border. J . Cell Biol., 101t2210-2222. Baron, R., L. Neff, P.T. Van, J.-R. Nefussi, and A. Vignery 1986 Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am. J . Pathol., 122:363 -378. Bar-Shavit, Z., S.L. Teitelbaum, P. Reitsma, A. Hall, L.E. Pegg, J . Trial. and A.J. Kahn 1983 Induction of monocvtic differentiation and bone resorption by 1,25-dihydroxyvitam:n D3. Proc. Natl. Acad. Sci. U.S.A., 80:5907-5911. Blair, H.C., A.J. Kahn, E.C. Crouch, J.J. Jeffrey, and S.L. Teitelbaum 1986 Isolated osteoclasts resorb the organic and inorganic components of bone. J . Cell Biol., 102:1164-1172. Bonucci, E. 1974 The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption. Calcif. Tiss. Res., 16:13-36. Bonucci, E. 1981 New knowledge on the origin, function and fate of osteoclasts. Clin. Orthop. Rel. Res., 158t252-269. Boyde, A., N.N. Ali, and S.J. Jones 1984 Resorption of dentine by isolated osteoclasts in vitro. Br. Dent. J., 156:216-220. Burger, E.H., J.W.M. van der Meer, J.S. van de Gevel, J.C. Gribnau, C.W. Thesingh, and R. van Furth 1982 In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes. J . Exp. Med., 156:1604-1614. Chambers, T.J. 1980 The cellular basis of bone resorption. Clin. Orthop. Rel. Res., 151:283-293. Chambers, T.J., K. Fuller, and J.A. Darby 1987 Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone. J. Cell. Physiol., 132:90-96. Doty, S.B., and B.H. Schofield 1972 Electron microscopic localization of hydrolytic enzymes in osteoclasts. Histochem. J., 4t245-258. Doty, S.B., and B.H. Schofield 1976 Enzyme histochemistry of bone and cartilage cells. Prog. Histochem. Cytochem., 8:l-37. Fallon, M.D. 1984 Bone resorbing fluid from osteoclasts is acidic: An in vitro micropuncture study. In: Endocrine Control of Bone and Calcium Metabolism, Vol. 8A. C.V. Cohn, T. Fujita, J.T. Potts, Jr., and R.V. Talmage, eds. Elsevier, Amsterdam, pp. 144-146. Gothlin, G., and L.E. Ericsson 1976 The osteoclast: Review of ultrastructure, origin and structure-function relationship. Clin. Orthop. Rel. Res., 12Ot201-231. Holtrop, M.E., and G.J. King 1977 The ultrastructure ofthe osteoclast and its function implications. Clin. Orthop. Rel. Res., 123: 177-196. Ibbotson, K.J., G.D. Roodman, L.M. McManus, and G.R. Mundy 1984 Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells. J . Cell Biol., 99:471-480. Kallio, D.M., P.R. Garant, and C. Minkin 1971 Evidence of coated 391 membranes in the ruffled border of the osteoclast. J . Ultrastruct Res., 37:169-177. Lucht, U. 1971 Acid phosphatase of osteoclasts demonstrated by electron microscopic histochemistry. Histochemie., 28: 103-117. MacDonald, B.R., N. Takahashi, L.M. McManus, J. Holahan, G.R. Mundy, and G.D. Roodman 1987 Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures. Endocrinology, 120:2326-2333. Minkin, C. 1982 Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif. Tissue Int., 34t285-290. Miyayama, H., R. Solomon, M. Sasaki, C. Lin, and W.H. Fishman 1975 Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique. J . Histochem. Cytochem., 23:439-451. Mundy, G.R., and G.D. Roodman 1987 Osteoclast ontogeny and function. In: Bone and Mineral Research, Vol. 5. William AP, ed. Elsevier, Amsterdam, pp. 209-279. Rath, N.C., A.R. Hand, and A.H. Reddi 1981 Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage, bone, and bone marrow development. Dev. Biol., 85:89-98. Roodman, G.D., K.J. Ibbotson, B.R. MacDonald, T.J. Kuehl, and G.R. Mundy 1985 1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow. Proc. Natl. Acad. Sci. U.S.A., 82:8213-8217. Sasaki, T., N. Motegi, H. Suzuki, C. Watanabe, K. Tadokoro, T. Yanagisawa, and S. Higashi 1988a Dentin resorption mediated by odontoclasts in physiologic root resorption of human deciduous teeth. Am. J . Anat., 183:303-315. Sasaki, T., N. Takahashi, H. Suzuki, C. Watanabe, S. Higashi, and T. Suda 198813Cytodifferentiation of odontoclasts in physiologic root resorption of human deciduous teeth. In: The Biological Mechanisms of Tooth Eruption and Root Resorption. 2. Daridoritch, ed. Ebsco Media, Birmingham, AL, pp. 321-328. Sasaki, T., A. Yamaguchi, S. Higashi, and S. Yoshiki 1985 Uptake of horseradish peroxidase by bone cells during endochondral bone development. Cell Tissue Res., 239t547-553. Silver, A,, R.J. Murrils, and D.J. Etherington 1988 Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell Res., 175t266-276. Takagi, N., R.T. Parmley, Y. Toda, and F.R. Denys 1982 Extracellular and intracellular digestion of complex carbohydrates by osteoclasts. Lab. Invest., 46:288-297. Takahashi, N., T. Akatsu, T. Sasaki, G.C. Nicholson, J.M. Moseley, T.J. Martin, and T. Suda 1988b Induction of calcitonin receptors by lu,25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells. Endocrinology, 123:1504-1510. Takahashi, N., H. Yamana, S. Yoshiki, G.D. Roodman, G.R. Mundy, S.J. Jones. A. Boyde, and T. Suda 1988a Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse marrow cultures. Endocrinology, 122:1373-1382. Walker, D.G. 1972 Enzymatic and electron microscopic analysis of isolated osteoclasts. Calcif. Tissue Res., 9:296-309. Warshawsky, H., D. Goltzman, M.F. Rouleau, and J.J.M. Bergeron 1980 Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat. J . Cell Biol., 85:682-694. van de Wijngaert, F.P., and E.H. Burger 1986 Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: A possible marker for (pre)osteoclast identification. J . Histochem. Cytochem., 34:13171323.