Defective bone remodelling in osteoprotegerin-deficient mice

Transcription

1 Japanese Society of Microscopy Journal of Electron Microscopy 52(6): (2003)... Full-length paper Defective bone remodelling in osteoprotegerin-deficient mice Norio Amizuka 1,5,*, Junko Shimomura 2,5, Minqi Li 1,3,5, Yukie Seki 1,3,5, Kimimitsu Oda 4,5, Janet E. Henderson 5,6, Atsuko Mizuno 7, Hidehiro Ozawa 8 and Takeyasu Maeda 1,5 1 Division of Oral Anatomy, 2 Division of Pediatric Dentistry, 3 Division of Oral and Maxillofacial Surgery and 4 Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Gakkochodori, Niigata , Japan, 5 Center for Transdisciplinary Research, Niigata University, Niigata , Japan, 6 Center for Bone and Periodontal Research, McGill University Health Centre, Royal Victoria Hospital, Montréal, Québec H3A 1A, Canada, 7 Department of Pharmacology, Jichi Medical University, Yakushiji , Japan and 8 Institute for Dental Science, Matsumoto Dental University, Shiojiri , Japan * To whom correspondence should be addressed. amizuka@dent.niigata-u.ac.jp... Abstract Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatasepositive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG / mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG / epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG / mice appear to cause bone fragility.... Keywords osteoclast, osteoblast, osteoprotegerin, bone remodelling, ultrastructure, bone matrix... Received 22 July 2003, accepted 25 August and bone [7]. Osteoprotegerin acts as a decoy receptor for RANKL, preventing its association with RANK and inhibiting osteoclastogenesis. Osteoprotegerin has an apparent molecular weight of 60 kda, is reported to act as a basic heparinbinding factor and has been identified as a disulphidelinked homodimer of 120 kda [8]. The role of OPG as a specific inhibitor of osteoclastogenesis has been confirmed by, e.g. transgenic mice with elevated circulating levels of OPG, overexpressed in liver, developed a severe osteopetrotic phenotype [9] and the exogenous administration of OPG rescued osteoporotic phenotype caused by ovariectomy [10]. In con- Introduction The receptor activator of NF B (RANK) is a member of the membrane-associated tumour necrosis factor (TNF) receptor family that plays a pivotal role in osteoclastogenesis [1,2]. RANK is expressed in osteoclast precursors whereas the RANK ligand (RANKL) is localized on the cell membrane of cells of the osteoblast lineage [1 3]. Cell cell contact between osteoblastic cells and osteoclast precursors brings RANK into contact with RANKL, thus, initiating osteoclastogenesis [4 6]. Osteoprotegerin (OPG) is a secretory product of cells in numerous tissues, including cartilage, intestine, lung, kidney, heart, skin

2 504 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 52, No. 6, 2003 trast, OPG-deficient mice developed osteopenia as a result of chronic osteoclastogenesis and increased bone resorption [11,12]. Thus, signalling through the OPG/RANK/RANKL axis is required for osteoclast formation and activity and for the regulation of bone resorption [6,13,14]. Bone formation is coupled to bone resorption during the process of remodelling, which continuously takes place along the surface of bone, with resorption preceding formation in both physiological and pathological circumstances. During the intermediate phase between resorption and formation, a reversal line is formed in the resorption lacuna, which becomes the cement line attaching new bone to the existing old bone surface [15]. Thus, cement lines are the histological hallmark of bone remodelling and their numbers and distribution are indicative of the rate and extent of bone turnover. Activation and progression of the sequence of cellular events that lead to bone remodelling is controlled at the level of cell cell and cell matrix interactions in the bone microenvironment. Osteopenia can result from uncoupling of the remodelling cycle, such that formation does not match resorption. The rapid decrease in bone volume in post-menopausal women results primarily from an increase in activation frequency caused by the rapid decline in circulating oestrogen. The slower decline that is seen in aging men and women is caused primarily by osteoblast senescence and secondary hyperparathyroidism, which leads to a reduction in the capacity of osteoblasts to adequately refill resorption lacunae. Paget s disease is a localized disorder of bone that is characterized by an initial increase in osteoclastogenesis followed by a compensatory increase in osteoblastogenesis and new bone formation. Recent work has confirmed that juvenile Paget s disease results from an OPG deficiency caused by homozygous deletion of the gene on chromosome 8q24.2 encoding OPG11B, a member of the superfamily of the TNF receptor (TNFRSF11B) [16]. Mice homozygous for targeted disruption of OPG are therefore a valid model to examine the pathogenesis of altered bone remodelling and skeletal fragility as seen in juvenile Paget s disease. However, few reports have demonstrated the histological features of osteoblastic activity and bone remodelling in OPG-deleted mice. Physiologically, the epiphysis is less subjected to bone remodelling than the metaphysis, which includes the site of endochondral bone formation, and, therefore, appears to be an adequate site for solely examining bone remodelling. In this study, we have attempted to analyse bone remodelling and the ultrastructure of the bone matrix of the tibial epiphyses in OPG-deficient mice in comparison with those of their wild-type littermates. Methods Tissue preparation All animal procedures were performed on 10-week-old male OPG / mice obtained as previously described [11] in accordance with guidelines for animal experimentation set by Niigata University. Mice were anaesthetized with an intraperitoneal injection of chloral hydrate and perfused through the left ventricle with either 4% paraformaldehyde diluted in 0.1 M phosphate buffer (ph 7.4) or a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in M cacodylate buffer (ph7.4). The femora and tibiae were dissected free of soft tissue and immersed in the same fixative for an additional 12 h at 4 C. After decalcification with 5% EDTA-2Na solution for 2 weeks at 4 C, some specimens were dehydrated through a graded series of ethanol prior to embedding in paraffin. Others were postfixed in a mixture of 1% osmium tetroxide and 1.5% potassium ferrocyanide for 4 h, dehydrated with ascending concentrations of acetone and embedded in epoxy resin (Taab, Berkshire, UK) prior to transmission electron microscope (TEM) observation (Hitachi H-7000; Hitachi Co. Ltd, Tokyo, Japan) at 80 kv. For dynamic labelling of mineralization, mice were injected with calcein (10 g/100 g body weight; Wako Pure Chemicals, Osaka, Japan) and sacrificed by cervical dislocation 24 h later [17]. Tibiae were cleaned and kept in 70% ethanol for 5 days at 4 C, stained according to Villaneuva [18,19] and dehydrated in graded ethanol prior to embedding in methyl metacrylate (Wako). Polymerized blocks were ground to the midpoint of the longitudinal axis and analysed using a confocal laser microscope (GB200; Olympus, Tokyo, Japan). Histochemistry for alkaline phosphatase, tartrateresistant acid phosphatase and osteopontin Five- m paraffin sections were used for alkaline phosphatase (ALP) and osteopontin immunohistochemistry and for tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry as previously reported [20]. Deparaffinized sections were treated with 0.1% hydrogen peroxidase for 15 min, to inhibit endogenous peroxidase, and pre-incubated with 1% bovine serum albumin in phosphate-buffered saline for 30 min at room temperature. Antisera against tissue non-specific ALP [21] or osteopontin (LSL Co., Tokyo, Japan) were applied to the sections at a dilution of 1:200 overnight at 4 C. Sections were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Chemicon International Inc., Temecula, CA, USA). Immune complexes were visualized using diaminobenzidine staining. The TRAP activity was detected by incubating with a mixture of 2.5 mg naphthol AS-BI phosphate (Sigma, St Louis, MO, USA), 18 mg red violet LB salt (Sigma) and 100 mm L (+) tartaric acid (0.76 g; Sigma) diluted in 0.1 M sodium acetate buffer (ph 5.0) for 15 min at 37 C. The sections were counterstained faintly with methyl green. Statistical analysis of TRAP-positive osteoclasts in the epiphyses The numbers of TRAP-positive osteoclasts in the entire region of the epiphyseal bone of five wild-type and five OPG / mice were counted. The TRAP-positive cells with more than two

3 N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice 505 Fig. 1 Lower magnified view of femora and tibiae of wild-type and OPG-deficient mice. The OPG-deleted femur (b) shows less-developed epiphyseal and metaphyseal trabecular bones when compared with the wild-type femur (a). The cartilaginous growth plate of the OPG / femur is penetrated by numerous connecting channels that form a continuum between the epiphyseal and metaphyseal bone, and divide the cartilage into islands (arrows) (b). Unlike the femur, however, the tibia of the OPG-deficient mouse (d) does not show striking alterations of metaphyseal trabeculae, but these are thinner and sparser when compared with those of the wild-type trabeculae (c). Original magnification: 25. Bars = 400 m.

4 506 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 52, No. 6, 2003 Fig. 2 Histological observation of tibial epiphyses of wild-type and OPG-deficient mice. The OPG / tibial epiphyses (b) are filled with numerous trabeculae similar to those of the wild-type mice (a). Epoxy resin sections ((c and e) wild-type, (d and f) OPG-deficient) show high resolutions of histological alterations seen in the OPG-deficient epiphysis. Although the intertrabecular region of the wild-type epiphysis is occupied with bone marrow (bm) (c), the corresponding intertrabecular area (asterisk) of the OPG-deficient epiphysis shows fibrous tissue (d). When observed at a higher magnification, flattened osteoblasts (ob) cover the surface of the wild-type epiphyseal trabeculae (c). On the contrary, the surface of the OPG-deleted epiphyseal trabeculae reveals well-formed osteoblasts and developed osteoclasts (oc) (f). Note that the soft tissue indicated by the asterisk in (f) includes fibroblastic cells. Abbreviation: GP, growth plate. Original magnification: (a, b) 60, (c, d) 280 and (e, f) 800. Bars = (a, b) 160 m, (c, d) 40 m and (e, f) 13 m. nuclei were regarded as osteoclasts. Results are expressed as means ± SE and statistical significance was evaluated using the Student s t-test. Results Morphological changes in tibial epiphyses of OPGdeficient mice The histological femoral sections of young adult OPG / mice exhibited a reduction in the number and size of trabecular bone spicules in both the metaphyses and epiphyses compared with their wild-type littermates (Figs 1a and 1b). In contrast to these changes in femoral architecture, the metaphyseal bone spicules of the OPG / tibia looked similar, although thinner and sparse, to those seen in wild-type mice (Figs 1c and 1d). The OPG / tibial epiphyses were filled with numerous trabeculae similar to those of the wild-type mice. At a higher magnification, however, the OPG / epiphyseal trabeculae were lined with numerous cuboidal osteoblasts (Figs 2b and 2d) compared with the flattened cells lining the surfaces of wild-

5 N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice 507 Fig. 3 The TRAP, ALP and osteopontin histochemistry on the tibial epiphyses. The tibial epiphyses of wild-type (a, c, e) and OPG-deficient (b, d, f) mice were subjected to enzyme histochemistry for TRAP (a, b) and immunostaining of ALP (c, d) and osteopontin (e, f). All insets demonstrate higher magnified images of each figure. A few flattened TRAP-positive osteoclasts (red) are detected in the wild-type mouse (a), whereas abundant TRAP-positive osteoclasts and cement lines (arrow in the inset) are formed in OPG-deficient epiphysis (b). In addition, thick cell layers of intense ALP-positive osteoblasts (ob; brown in the inset) occupy the intertrabecular region of OPG-depleted epiphysis (d). However, a thin cell layer of ALP-positive cells covers the trabecular surfaces in the wild-type counterpart (c). A fine meshwork of numerous osteopontin-positive cement lines (brown in the inset) form in the OPG-deficient epiphysis (f), whereas the wild-type trabeculae reveal only a few osteopontin-positive cement lines (e). Note the distribution of osteopontin-immunopositive cement lines of the OPG-deficient mouse manifest correspondence with TRAP-positive cement lines, as shown in (b). Original magnification: (a f) 50 and (insets) 120. Bars = 40 m. type trabeculae (Figs 2a and 2c). Semi-thin sections imaged at a higher resolution revealed that cuboidal osteoblasts adjacent to the bone surfaces and well-developed preosteoblasts occupied much of the marrow cavity in OPG / mice (Fig. 2f) when compared with the wild-type counterpart (Fig.2e). Large osteoclasts were frequently located on the epiphyseal trabeculae of OPG / mice. Osteoclast and osteoblast activity and bone remodelling It therefore appeared that the increase in osteoclast formation in OPG / mice gave rise to a coupled increase in mature osteoblasts. To investigate the activity of catabolic and anabolic cells in the tibial epiphysis, we performed enzyme histochemistry for TRAP and immunostaining for ALP, which are recognized markers for osteoclast and osteoblast activity, respectively. In the absence of OPG, numerous TRAP-positive osteoclasts were seen along the trabecular surfaces of OPG /

6 508 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 52, No. 6, 2003 Fig. 4 Confocal laser microscopic images of calcein deposition in the tibial epiphyses. Lower magnified images show abundant labelling with calcein (yellow or green) in the OPG-deleted tibia (b) compared with the wild-type littermate (a). The specimens were counterstained with Villaneuva dye (see Methods ). When examined under higher magnification, the intense and numerous distribution of calcein can be seen in the OPG-deficient epiphysis (d) compared with their wild-type littermate (c). Abbreviations: GP, growth plate; epi, epiphysis; meta, metaphysis. Original magnification: (a, b) 36 and (c, d) 70. Bars = (a, b) 280 m and (c, d) 140 m. bone compared with that of their wild-type littermates (Figs 3a and 3b). Statistical analysis showed a significant increase in the osteoclast number in the epiphyses of the OPG / mice (42.80 ± 5.40, P < 0.005) when compared with those from the wild-type mice (7.80 ± 2.17). Substantial TRAP staining was also seen deposited along the numerous cement lines in OPG / bone, but not in control bone (compare Figs 3a and 3b). The cell layer of preosteoblasts and cuboidal osteoblasts lining the epiphyseal trabeculae of OPG / mice stained intensely for ALP compared with the more discrete staining seen in the wild-type mice (Figs 3c and 3d). In addition to TRAP, the fine meshwork of cement lines seen in the mutant bone also stained heavily for osteopontin, which is known to mediate osteoclast attachment to the bone surface (Figs 3e and 3f). The increase in the number and activity of osteoblasts lining the epiphyseal trabeculae was reflected in the striking increase in calcein deposited at the mineralization fronts in the epiphyses of OPG / mice compared with wild-type littermates (Fig. 4a d).

7 N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice 509 Fig. 5 Ultrastructural observations in bone matrix of the wild-type epiphysis. The wild-type bone matrix exhibited a smooth-contoured surface covered with osteoblasts (ob) (a). When observed at a higher magnification (b), bone matrix is composed of densely connected collagen fibres. The cement line is recognized as an osmiophilic thin line (white arrows). The upper inset shows collagen fibres relatively sparse in the superficial portion of the trabeculae (inset, a), whereas in the inner portion, densely packed collagen fibres are discernible (inset, b). Note little space among the condensed collagen fibres. Abbreviation: ocy, osteocyte. Original magnification: (a) 1400, (b) 6000 and (insets) Bars = (a) 7 m and (b) 1.5 m. Ultrastructural analysis of bone matrix in OPGdeficient mice Ultrastructural analysis of wild-type epiphyses using TEM revealed mature osteoblasts adjacent to smooth-contoured trabecular surfaces (Fig. 5) compared with the irregular surfaces seen in OPG / bone (Fig. 6). The dense, highly organized matrix with densely packed bundles of collagen fibres in wildtype mice was in contrast to the disorganized matrix with sparse, randomly oriented collagen fibrils interspersed among abundant amorphous organic material of OPG / epiphyses (compare Figs 5 and 6). The cement lines in wild-type mice were observed as thin, osmiophilic lines connecting adjacent bone matrices, whereas those in OPG / bone were thick, translucent, often discontinuous and randomly oriented (Fig. 6b). At a higher resolution, the cement lines in wild-type bones were seen to consist of a prominent osmiophilic line

8 510 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 52, No. 6, 2003 Fig. 6 Ultrastructural observations in the bone matrix of the OPG-deleted epiphysis. The OPG-deficient trabecular bone shows an irregularly shaped surface covered with cuboidal osteoblasts (ob) (a). Cement lines with concaved shape (white arrows) can be numerously observed. At higher magnification, a cement line (black arrowheads) can be seen to run across another cement line (white arrows) (b). Abbreviations: ob, osteoblast; ocy, osteocyte. Original magnification: (a) 1400 and (b) Bars = (a) 7 m and (b) 1 m. adjacent to a less prominent translucent zone (Fig. 7a) whereas in OPG / bone the osmiophilic line was reduced in thickness and the translucent zone greatly expanded (Fig. 7b). These broad translucent zones were often seen to terminate in fissures (Fig. 7c). Discussion In our study, increased osteoclast formation and activation were accompanied by accelerated osteoblastic activities in the OPG-deficient tibial epiphyses, though wild-type epiphyses showed slow turnover of bone. In this mutant mouse, an area of focus was the abundant ALP-positive osteoblastic cell, which occupied the intertrabecular region (Figs 2d and 2f). Therefore, activated osteoclasts and/or some factor released from newly resorbed bone matrix may affect cell proliferation and subsequent differentiation of osteoblastic lineage cells. Consistent with this hypothesis was the presence of abundant calcein deposition in the OPG / mice, indicating that bone mineralization was enhanced and osteoblast activity must have been increased. In general, cellular coupling is achieved by sequential events: once the phase of osteoclastic resorption is completed, osteoblastic cells localize to the previous resorption surface and deposit bone matrices, which give rise to cement lines where new bone attaches to the pre-existing old bone surface. Although, under physiological circumstances, the epiphysis is less subject to bone remodelling, numerous complex meshworks of cement lines were seen in the OPG /

9 N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice 511 Fig. 7 Highly magnified images of cement lines of the wild-type and the OPG-deficient bone matrices. Under higher magnification, in the inner portion of the wild-type bone, a cement line is composed of an osmiophilic thin linear structure (arrows) that closely attached bone matrices (a). Note a narrow translucent area underlies the osmiophilic linear structures. In contrast to the wild-type bone, in OPG-deficient mice a faint and much thinner osmiophilic linear structure (arrows) is seen accompanied with the relative thick translucent area (arrows) (b). The OPG-deficient bone matrix included sparse collagen fibres with irregular directions. In some portions, cement lines with concaved shape (white arrowheads) are seen (c). A translucent fissure with a large thickness terminates in a fissure corresponding to a micro-fracture (double white arrows). The inset exhibits a higher magnification of the separation of bone matrices in the fissure. Original magnification: (a, b) , (c) 6000 and (inset) Bars = (a, b) 1 m and (c) 1.5 m.

10 512 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 52, No. 6, 2003 epiphyses, indicating that bone remodelling was markedly accelerated. This accelerated bone remodelling gave rise to a woven bone matrix, which would be more easily resorbed. Thus, OPG deficiency not only stimulated bone remodelling but also contributed to a decline in the quality of bone matrix. Therefore, it is of significance to investigate bone matrices of this mutant mouse since the bone quality, including strengthbearing mechanical stress, stiffness, rigidity and flexibility, is mainly attributed to the properties of the bone matrices. The OPG / mice showed sparse collagen fibres with abundant organic components, which are characteristic properties of woven bone, while the wild-type counterparts displayed densely packed collagen bundles in a preferential direction. Biomechanical properties, such as strength and toughness, must derive from its solid-phase components, and ultimate yield strength is determined by both mineral composition and by the integrity of the collagen in bone [22]. The mechanical properties that collagen fibres provide to bone have been verified by the use of mice heterozygous for type-i collagen gene deletion [23,24]. As a consequence, the reduced synthesis of type-i collagen led to a reduction in stiffness and strength properties under static loading. Consistently, the OPG-deficient bone with sparse collagen fibrils may show diminished stiffness and strength against mechanical loading. The speed of bone turnover appears to be involved in the mechanical strength of bone. If bone matrix undergoes rapid degradation and repair, e.g. high bone turnover, it will show reduced mechanical stiffness and strength relative to intact bone matrix. Thus, bone that is in a state of high turnover, including in Paget s disease, might give rise to diminution in mechanical properties when compared with bone subjected to normal remodelling. It is of importance to recognize that this phenomenon occurs independently of bone density. Thus, even though bone strength is correlated with density, the remodelling state of bone may be a more important factor with respect to the risk of fracture. Although it is obvious that the interfacial structure of cement lines is of paramount importance [25], the precise mechanisms of cement lines involved in cell-to-matrix and matrix-to-matrix interactions are still being discussed. The cement lines contain a larger amount of organic components, e.g. local factors and bone matrix proteins, including osteopontin [26 29]. A physiologically formed cement line, under balanced remodelling between bone resorption and formation, appears to show optimal amounts of organic components and close attachment of bone matrices, presumably, enabling formation of ridged matrix-adhesion at this site. In the case of OPG / bone, however, cement lines with a markedly enlarged translucent zone often terminate in fissures. These histological findings imply that the OPG deficiency not only impairs bone remodelling but also lessens the quality of the bone matrix. Additionally, the meshwork of cement lines across osteocytic cytoplasmic processes may disrupt the interconnections of osteocytes/osteoblasts. Cellular activities play an important role in determining the mechanical properties of bone. For instance, the viscoelasticity of bone is largely owing to its water content [30], which appears to be mostly regulated by osteocytes and osteoblasts. In patients with juvenile Paget s disease, the continual rapid formation and degradation of osseous tissue result in impaired growth, modelling and remodelling of the entire skeleton. Recently, juvenile Paget s disease has been reported to derive from a deficiency of the OPG gene [16]. Like juvenile Paget s disease, OPG / mice showed specific properties with rapid bone remodelling, histologically fragile structures with numerous thick cement lines and bone matrices with sparse collagen fibres. The findings seen in the OPG / mouse might, therefore, contribute to our understanding of the pathological features of bone in juvenile Paget s disease, which include deterioration of architectural integrity as a result of generalized and local rapid remodelling of bone. Concluding remarks In OPG-deficient mice, osteoblastic activity and resultant bone remodelling are stimulated in conjunction with enhanced osteoclastic bone resorption. This high rate of bone turnover results in poor attachment of histologically fragile bone matrices at the cement lines, thereby contributing to poor bone quality. Acknowledgements This work was supported by grants from the Promotion of Niigata University Research Project and the Ministry of Culture, Education, Science Sports and Technology of Japan, and an award from the academic meeting of Osteoporosis Japan. References 1 Lacey D L, Timms E, Tan H L, Kelley M J, Dunstan C R, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian Y X, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, and Boyle W J (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93: Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, and Suda T (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95: Tsukii K, Shima N, Mochizuki S, Yamaguchi K, Kinosaki M, Yano K, Shibata O, Udagawa N, Yasuda H, Suda T, and Higashio K (1998) Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem. Biophys. Res. Commun. 246: Takahashi N, Udagawa N, Akatsu T, Tanaka H, Isogai Y, and Suda T (1991) Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128: Amizuka N, Takahashi N, Udagawa N, Suda T, and Ozawa H (1997) An ultrastructural study of cell cell contact between mouse spleen cells and calvaria-derived osteoblastic cells in a co-culture system for osteoclast formation. Acta Histochem. Cytochem. 30:

11 N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie M T, and Martin T J (1999) Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocrin. Rev. 20: Simonet W S, Lacey D L, Dunstan C R, Kelley M, Chang M S, Luthy R, Nguyen H Q, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan H L, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes T M, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, and Boyle W J (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89: Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, and Higashio K (1997) Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem. Biophys. Res. Commun. 234: Min H, Morony S, Sarosi I, Dunstan C R, Capparelli C, Scully S, Van G, Kaufman S, Kostenuik P J, Lacey D L, Boyle W J, and Simonet W S (2000) Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J. Exp. Med. 192: Shimizu-Ishiura M, Kawana F, and Sasaki T (2002) Osteoprotegerin administration reduces femural bone loss in ovariectomized mice via impairment of osteoclast structure and function. J. Electron Microsc. 51: Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, and Ozawa H (1998) Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem. Biophys. Res. Commun. 247: Bucay N, Sarosi I, Dunstan C R, Morony S, Tarpley J, Capparelli C, Scully S, Tan H L, Xu W, Lacey D L, Boyle W J, and Simonet W S (1998) Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 12: Hofbauer L C, Khosla S, Dunstan C R, Lacey D L, Boyle W J, and Riggs B L (2000) The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J. Bone Miner. Res. 15: Khosla S (2001) The OPG/RANKL/RANK system. Endocrinology 142: Baron R (1999) Anatomy and ultrastructure of bone. In: Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, ed. Favus M J, pp. 3 10, (Lippincott Williams & Wilkins, Philadelphia). 16 Whyte M P, Obrecht S E, Finnegan P M, Jones J L, Podgornik M N, McAlister W H, and Mumm S (2002) Osteoprotegerin deficiency and juvenile Paget s disease. N. Engl. J. Med. 347: Amizuka N, Karaplis A C, Henderson J E, Warshawsky H, Lipman M L, Matsuki Y, Ejiri S, Tanaka M, Izumi N, Ozawa H, and Goltzman D (1996) Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev. Biol. 175: Villanueva A R (1974) A bone stain for osteoid seams in fresh, unembedded, mineralized sections of bone. Stain Technol. 49: Villanueva A R and Lundin K D (1989) A versatile new mineralized bone stain for simultaneous assessment of tetracycline and osteoid seams. Stain Technol. 64: Amizuka N, Yamada M, Watanabe J, Hoshi K, Fukushi M, Oda K, Ikehara Y, and Ozawa H (1998) Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow. Microsc. Res. Tech. 41: Oda K, Amaya Y, Fukushi-Irie M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S, and Kobayashi J (1999) A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissuenonspecific alkaline phosphatase. J. Biochem. 126: Burstein A, Zika J C, Heiple K G, and Klein L (1977) Contribution of collagen and mineral to the elastic plastic properties of bone. J. Bone J. Surg. 57A: Bonadio J, Saunders T L, Tsai E, Goldstein S A, Morris-Wiman J, Brinkley L, Dolan D F, Altschuler R A, Hawkins J E Jr, Bateman J F, Mascara T, and Jaenisch R (1990) Transgenic mouse model of the mild dominant form of osteogenesis imperfecta. Proc. Natl. Acad. Sci. USA 87: Bonadio J, Jepsen K J, Mansoura M K, Jaenisch R, Kuhn J L, and Goldstein S A (1993) A murine skeletal adaptation that significantly increases cortical bone mechanical properties. Implications for human skeletal fragility. J. Clin. Invest. 92: Burr D B, Schaffler M B, and Frederickson R G (1988) Composition of the cement line and its possible mechanical role as a local interface in human compact bone. J. Biochem. 21: McKee M D, Farach-Carson M C, Butler W T, Hauschka P V, and Nanci A (1993) Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HSglycoprotein) proteins in rat bone. J. Bone Miner. Res. 8: McKee M D and Nanci A (1996) Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microsc. Res. Tech. 33: McKee M D and Nanci A (1996) Osteopontin: an interfacial extracellular matrix protein in mineralized tissues. Connect. Tissue Res. 35: McKee M D and Nanci A (1996) Osteopontin deposition in remodeling bone: an osteoblast mediated event. J. Bone Miner. Res. 11: Cullinane D M and Einhorn T A (2002) Biomechanics of Bone. In: Principles of Bone Biology, eds Bilezikian J P, Raisz L G, and Rodan G A, pp , (Academic Press, New York).