J Electron Microsc (Tokyo) 2003 Amizuka 503 13

8/12/2019 J Electron Microsc (Tokyo) 2003 Amizuka 503 13

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Journal of Electron Microscopy 52(6): 503513 (2003) Japanese Society of Microscopy

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Full length paperDefective bone remodelling in osteoprotegerin-deficientmice

Norio Amizuka1,5,*, Junko Shimomura2,5, Minqi Li1,3,5, Yukie Seki1,3,5, Kimimitsu Oda4,5, Janet

E. Henderson5,6, Atsuko Mizuno7, Hidehiro Ozawa8and Takeyasu Maeda1,5

1Division of Oral Anatomy, 2Division of Pediatric Dentistry, 3Division of Oral and Maxillofacial Surgery and4Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-

dori, Niigata 951-8514, Japan, 5Center for Transdisciplinary Research, Niigata University, Niigata 951-8514, Japan,6Center for Bone and Periodontal Research, McGill University Health Centre, Royal Victoria Hospital, Montral,

Qubec H3A 1A, Canada, 7Department of Pharmacology, Jichi Medical University, Yakushiji 329-0498, Japan and8Institute for Dental Science, Matsumoto Dental University, Shiojiri 399-0704, Japan*To whom correspondence should be addressed. E-mail: [email protected]

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Abstract Previous studies have reported enhanced osteoclastogenesis, increasedbone 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 phosphatase-

positive 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 phos-

phatase 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. Intotal, 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..................................................................................................................................................................................................................

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Received 22 July 2003, accepted 25 August 2003

Introduction

The receptor activator of NFB (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 [13]. Cellcell contact between osteo-

blastic cells and osteoclast precursors brings RANK into contact

with RANKL, thus, initiating osteoclastogenesis [46]. Osteo-

protegerin (OPG) is a secretory product of cells in numerous

tissues, including cartilage, intestine, lung, kidney, heart, skin

and bone [7]. Osteoprotegerin acts as a decoy receptor for

RANKL, preventing its association with RANK and inhibiting

osteoclastogenesis. Osteoprotegerin has an apparent molecu-

lar weight of 60 kDa, is reported to act as a basic heparin-

binding factor and has been identified as a disulphide-linked 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 pheno-

type [9] and the exogenous administration of OPG rescued

osteoporotic phenotype caused by ovariectomy [10]. In con-


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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, 2003504

trast,OPG-deficientmicedevelopedosteopeniaasaresultof 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 inbothphysiologicalandpathologicalcircumstances.Duringthe 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 distribu-

tion 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 cellmatrix interactions in the bone microenviron-

ment.

Osteopenia can result from uncoupling of the remodelling

cycle, such that formation does not match resorption. Therapid 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 hyper-

parathyroidism, which leads to a reduction in the capacity of

osteoblasts to adequately refill resorption lacunae. Pagets

disease is a localized disorder of bone that is characterized by

an initial increase in osteoclastogenesis followed by a com-

pensatory increase in osteoblastogenesis and new bone forma-

tion. Recent work has confirmed that juvenile Pagets disease

results from an OPG deficiency caused by homozygous dele-

tion 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 there-

fore a valid model to examine the pathogenesis of altered bone

remodelling and skeletal fragility as seen in juvenile Pagets

disease. However, few reports have demonstrated the histolog-

ical 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, there-

fore, appears to be an adequate site for solely examining bone

remodelling. In this study, we have attempted to analyse boneremodelling 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 accord-

ance with guidelines for animal experimentation set by

Niigata University. Mice were anaesthetized with an intra-

peritoneal injection of chloral hydrate and perfused through

the left ventricle with either 4% paraformaldehyde diluted in

0.1 Mphosphate buffer (pH 7.4) or a solution of 2% parafor-

maldehyde and 2.5% glutaraldehyde in 0.067 M cacodylate

buffer (pH7.4). The femora and tibiae were dissected free of

softtissueand immersed inthesamefixativeforanaddi-tional 12 h at 4C. After decalcification with 5% EDTA-2Nasolution for 2 weeks at 4C, 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 elec-

tron microscope (TEM) observation (Hitachi H-7000; Hitachi

Co. Ltd, Tokyo, Japan) at 80 kV.

For dynamic labelling of mineralization, mice were injected

with calcein (10g/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

4C, 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, tartrate-

resistant acid phosphatase and osteopontin

Five-m paraffin sections were used for alkaline phosphatase

(ALP) and osteopontin immunohistochemistry and for tar-

trate-resistant acid phosphatase (TRAP) enzyme histochemis-

try as previously reported [20]. Deparaffinized sections weretreated 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 4C. Sections

were then incubated with horseradish peroxidase-conjugated

goat anti-rabbit IgG (Chemicon International Inc., Temecula,

CA, USA). Immune complexes were visualized using diamino-

benzidine staining.

The TRAP activity was detected by incubating with a mix-

ture of 2.5 mg naphthol AS-BI phosphate (Sigma, St Louis,

MO, USA), 18 mg red violet LB salt (Sigma) and 100 mML (+)

tartaric acid (0.76 g; Sigma) diluted in 0.1 Msodium acetate

buffer (pH 5.0) for 15 min at 37C. The sections were counter-

stained 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


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N. Amizuka et al. Bone remodelling in osteoprotegerin-deficient mice 505

Fig. 1Lower magnified view of femora and tibiae of wild-type and OPG-deficient mice. The OPG-deleted femur (b) shows less-developed epi-

physeal 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.


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nuclei were regarded as osteoclasts. Results are expressed asmeans SE and statistical significance was evaluated using

the Students t-test.

Results

Morphological changes in tibial epiphyses of OPG-

deficient 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 comparedwith 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 thin-

ner 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-

Fig. 2Histological 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.


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typetrabeculae(Figs2aand2c).Semi-thinsectionsimagedat a higher resolution revealed that cuboidal osteoblasts adja-

cent 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 ana-

bolic cells in the tibial epiphysis, we performed enzyme histo-

chemistry 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/

Fig. 3The 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 abun-

dant 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-pos-

itive 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: (af) 50 and (insets) 120. Bars = 40 m.


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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 epi-

physes of OPG/mice compared with wild-type littermates

(Fig. 4ad).

Fig. 4Confocal 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

Villaneuvadye(seeMethods).Whenexaminedunderhighermagnification,theintenseandnumerousdistributionofcalceincanbeseenin the OPG-deficient epiphysis (d) compared with their wild-type littermate (c). Abbreviations: GP, growth plate; epi, epiphysis; meta, meta-

physis. Original magnification: (a, b) 36 and (c, d) 70. Bars = (a, b) 280 m and (c, d) 140 m.


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Ultrastructural analysis of bone matrix in OPG-

deficient mice

Ultrastructural analysis of wild-type epiphyses using TEM

revealed mature osteoblasts adjacent to smooth-contoured

trabecular surfaces (Fig. 5) compared with the irregular sur-

faces seen in OPG/bone (Fig. 6). The dense, highly organized

matrix with densely packed bundles of collagen fibres in wild-

type 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

Fig. 5Ultrastructural 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) 24 000. Bars = (a) 7

m and (b) 1.5 m.


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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 epiphysesshowedslowturnoverofbone.Inthismutantmouse,anarea 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 resorp-

tion 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/

Fig. 6Ultrastructural 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) 9000. Bars = (a) 7 m and (b) 1 m.


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Fig. 7Highly 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) 12 000, (c) 6000 and

(inset) 12 000. Bars = (a, b) 1 m and (c) 1.5 m.


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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 strength-

bearing mechanical stress, stiffness, rigidity and flexibility, ismainly attributed to the properties of the bone matrices. The

OPG/ mice showed sparse collagen fibres with abundantorganic 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 veri-

fied 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-defi-

cient 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 Pagets 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 osteo-

pontin [2629]. 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 for-

mation 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 histologi-

cal 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 interconnec-

tions 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 regu-

lated by osteocytes and osteoblasts.

In patients with juvenile Pagets disease, the continual rapid

formation and degradation of osseous tissue result in impaired

growth, modelling and remodelling of the entire skeleton.

Recently, juvenile Pagets disease has been reported to derive

from a deficiency of the OPG gene [16]. Like juvenile Pagetsdisease, OPG/ mice showed specific properties with rapidbone 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 Pagets disease, which include

deterioration of architectural integrity as a result of general-

ized 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 Univer-

sity Research Project and the Ministry of Culture, Education, Science

Sports and Technology of Japan, and an award from the academic meeting

of Osteoporosis Japan.

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