Advanced Glycation End Products Enhance Osteoclast-

Induced Bone Resorption in Cultured Mouse Unfractionated

Bone Cells and in Rats Implanted Subcutaneously with
Devitalized Bone Particles

TOSHIO MIYATA,* KOHEI NOTOYA,t KEIJI YOSHIDA,t KATSUNORI HORIE,

KENJI MAEDA, KIYOSHI KUROKAWA,* and SHIGEHISA TAKETOMIt
*Jnstitute of Medical Sciences and Department of Medicine, Tokai University School of Medicine, Isehara,

Japan; tPharmaceutical Research Laboratories II, Pharmaceutical Research Division, Takeda Chemical
industries, Osaka, Japan; Department of internal Medicine, Branch Hospital, Nagoya University School of
Medicine, Nagoya, Japan.

Abstract. Advanced glycation end products (AGE) are formed bone resorption was effectively inhibited by calcitonin and
in long-lived matrix proteins by a nonenzymatic reaction with ipriflavone, both of which are inhibitors of bone resorption.
sugar. The presence of AGE in j32-microgbobulin-amyboid AGE-enhanced bone resorption was further supported by in
fibrils of dialysis-rebated amyboidosis, one of the characteristic vivo evidence that rat bone particles-upon incubation with
features of which is an accelerated bone resorption around glucose for 60 days (AGE-bone particbes)-when implanted
amyboid deposits, was recently demonstrated. This suggested a subcutaneously in rats, were resorbed to a much greater extent
potential link of AGE in bone resorption and initiated this than control bone particles upon parallel incubation without
investigation of whether AGE enhance bone resorption. When glucose. These findings suggest that AGE enhance osteoclast-
mouse unfractionated bone cells containing osteoclasts were induced bone resorption. Although the mechanism remains
cultured on dentin slices, both AGE-modified /32-microgbobu- unknown, AGE are unlikely to promote differentiation of os-
bin and BSA increased the number of resorption pits formed by teoclast progenitors into osteoclasts, suggesting that AGE ac-
osteoclasts, whereas their normal counterparts or those modi- tivate osteoclasts or alter microenvironments favorable for
fled with the early glycation products did not. AGE proteins, bone resorption by osteoclasts. The modification of bone ma-
however, did not increase the number of newly formed oste- trices with AGE might play a robe in the remodeling of senes-
oclasts, even in the coculture of mouse bone marrow cells with cent bone matrix tissues, further implicating a pathological
osteoblastic cells isolated from mouse calvaria. Enhanced bone significance of AGE in dialysis-related amyboidosis or osteo-
resorption was also observed when unfractionated bone cells porosis associated with diabetes and aging. (J Am Soc Nephrob
were cultured on AGE-modified dentin slices. AGE-enhanced 8: 260-270, 1997)

Advanced glycation end products (AGE) are the pigmented proteins (2), by the stimulation of cellular responses (3- 8) via
and fluorescent adducts that are formed, over the months, by a receptors specific for AGE proteins (9,10), or by the generation
non-enzymatic reaction between sugar and long-lived proteins of reactive oxygen intermediates (1 1 , 12). AGE have thus been
such as matrix coblagens, cabled the Maillard reaction (1). implicated in the pathogenesis of atherosclerosis (3,4,13), di-
Several lines of evidence have suggested that AGE proteins abetic nephropathy (5,6), dialysis-related amyboidosis (7,8,1 4-
play a role in normal tissue remodeling, i.e., the removal and 17), and Alzheimer’s disease (18-20).
replacement of senescent extraceblular matrix components (2). Recently, we demonstrated that AGE are present in amyboid

However, under pathological conditions such as diabetes, renal fibribs of dialysis-rebated amyboidosis (14,15). This is a serious
failure, and aging, the accumulation of AGE proteins might osteoarticubar complication of bong-term hemodiabysis patients,

lead to tissue damage through a variety of mechanisms: in which amyboid fibrils consisting predominantly of p2-mi-
through an alteration of the structure and function of tissue crogbobubin (j32m) deposit particularly in periarticubar bones
(21 ,22). One of the characteristic features of this complication
is an accelerated bone resorption around amyboid deposits,
which beads to subchondrab erosion and bone cysts-detected
Received May 21, 1996. Accepted August 12, 1996. by radiological examination-in most patients undergoing he-
Correspondence to Dr. Toshio Miyata, Institute of Medical Sciences and
modialysis for more than 10 yr (21,22). Further studies in our
Department of Medicine, Tokai University School of Medicine, Bohseidai,
Isehara, Kanagawa 259- 1 1 , Japan.
group demonstrated that the j32m modified with AGE (AGE-

1046-6673/0802-0260$03.0O/O 132m), but not intact 32m, induces chemotaxis of monocytes

Journal of the American Society of Nephrology and secretion of bone-resorbing cytokines such as interleukin
Copyright © 1997 by the American Society of Nephrology (IL)- 1/3, tumor necrosis factor (TNF)-a, and IL-6 from mono-
 AGE Enhance Osteoclast-Induced Bone Resorption 261

cyte-derived macrophages (7,8,17). These findings suggest a Removal of Endotoxin

potential link of AGE to bone resorption. To perform the experiments in endotoxin-free materials, we care-
The AGE levels of matrix proteins have been shown to be fully removed endotoxin from the proteins by incubating them with
markedly elevated in diabetic patients and elderly subjects endotoxin-adsorbent (Pyro SepR; Daicel Chemical Industries, Ltd.,
when compared with healthy young subjects (23,24). Taking Tokyo, Japan) for 2 h at 4#{176}C, followed by filtration through a 0.22
gm-pore filter (Ultrafree C3-GV: Nihon Milbipore Ltd., Tokyo, Ja-
into account the observations that the AGE modification accu-
pan) to remove the adsorbent. The endotoxin bevels were measured by
mulates in long-lived proteins in vivo (1,2) and that among
a kit (Toxicolor#{174} System; Seikagaku Corp., Tokyo, Japan), and all the
these are bone matrix proteins such as Type I collagen, we
materials and media used in this study were found to be endotoxin-
assumed that AGE might play a pathophysiological role not free (<0.5 U of endotoxinlmL).
only in dialysis-related amyboidosis, but also in the remodeling
of senescent bone matrix tissues or osteoporosis associated Unfractionated Bone Cell Preparation
with diabetes and aging. In the study presented here, we Unfractionated bone cells were prepared according to the method
examined the effect of AGE on osteoclast-induced bone re- of Takada et al. (28). In brief, the femur and tibia of 1 1- to 13-day-old
sorption in vitro utilizing the pit-formation assay with an ICR mice (Japan Charles River, Tokyo, Japan) were aseptically iso-
unfractionated bone cell culture system containing osteoclasts lated and minced with scalpel blades into 20 mL of a culture medium
consisting of a-Minimum Essential Medium (a-MEM) containing 5%
from newborn mice. Because bone resorption is regulated by
fetal bovine serum (FBS), 10 mM HEPES (pH 7.0), 100 .tWmL
two different processes, i.e., differentiation of osteoclast pro-
kanamycin, and 80 g/mL gentamicin. The mixture of cell suspension
genitors into osteoclasts and activation of osteoclasts (25), we
and bone fragments were gently pipeued for 5 mm and allowed to
examined the effect of AGE on osteoclast formation by means stand for S mm. The resulting supernatant was used for the experi-
of the mouse coculture system of bone marrow cells with ments.
primary osteoblastic cells. An inhibitory effect of clinically To determine the number of osteoclasts in the supematant, an
used anti-bone resorbing agents on bone resorption enhanced aliquot was smeared on a slide glass and stained for tartrate-resistant
by AGE was also evaluated. Furthermore, we investigated the acid phosphatase (TRAP), a marker enzyme for osteoclasts, according
bone-resorbing effect of AGE in vivo in rats that were im- to the method of Burstone (29). TRAP-positive multinucbeated cells
planted subcutaneously with bone particles. (MNC) with three or more nuclei (roughly corresponding to 0.05% of
the total cells) were counted as osteoclast-like cells.

Pit-Formation Assay
Materials and Methods Dentin slices with 0. 1 mm-thickness and 6 mm-diameter were
Both normal and electrophoretically acidic isoforms of 32m were prepared by our previous method (30) and placed in a 96-well culture
purified from the urine of nondiabetic hemodialysis patients who were dish (NUNCLON; Nunc, Roskibide, Denmark). The unfractionated
free of urinary infection as described previously (14). Our previous bone cell suspension (4 X b0 cells/dentin slice) with a TRAP-
study demonstrated that the acidic 32m, but not normal (32m, had the positive MNC density of 200 cells/dentin slice was seeded on to
characteristic physicochemical properties of AGE proteins, i.e., brown dentin slices and allowed to stand for 2 h at 37#{176}C
in a CO2 incubator.
color, fluorescence, and tendency for polymerization, and exhibited After removal of nonadherent cells, the cells were further cultured for
positive immunoreactivity to anti-AGE antibody ( 14). The levels of 4 days in the medium containing several concentrations of either
pentosidine (26), which is a glycoxidation marker postulated to be AGE-j32m or normal 2m, or b0 M la, 25-dihydroxyvitamin D3
AGE (1 1), were 2.12 mmol/mol in acidic 32m and below the detection (la, 25(OH)2D3). In some experiments, the cells were cultured on
limit (0.02 mmol/mob) in normal (32m, assessed by HPLC assay dentin slices with or without bO_6 M in vitro-prepared AGE-2m in
according to the method by Odetti et al. (27). This also supports that the presence of bO_8 M chick calcitonin (Peninsula Laboratories Co.,
AGE are present in the acidic 2m, but not in normal 2m. Belmont, CA) or l0 M ipriflavone (7-isopropoxyisoflavone, syn-
Proteins modified with the Mailbard products such as AGE or thesized at Takeda Chemical Industries, Osaka, Japan). Experiments
Amadon products, the early Maillard products leading to AGE, were were completed by removing the culture medium and adding 0. 1 M
also prepared in vitro. Five hundred micrograms of normal human cacodylate buffer solution (pH 7.4) containing 2% paraformaldehyde.
2m or BSA (essentially fatty acid-free grade; Sigma, St. Louis, MO) Total number of TRAP-positive MNC on each dentin slice was
was incubated with 0.1 M D-gbucOse (Wako Pure Chemicals, Osaka, counted after TRAP staining of the cells. Cells were then removed
Japan) at 37#{176}C
for 60 days or 10 days in 500 L of 0. 1 M phosphate from dentin slices by ubtrasonication for 30 s in distilled water, and
buffer (pH 7.4) containing 1.5 mM phenybmethanesulfonyl fluoride air-dried. The slices were stained with hematoxylin and the number of
(PMSF; Sigma) under sterile conditions. The presence of AGE in densely stained pits was counted under light microscopy.
glycated 32m after a 60-day incubation and the presence of Amadori In some experiments, dentin slices were preincubated with or
products in glycated (32m after a 10-day incubation were shown without 0.5 M glucose in 20 mL of 0.3 M phosphate buffer (pH 7.4)
previously (14-17). The pentosidine levels, determined by HPLC at 37#{176}C
for 40 days. The presence of AGE in the glycated dentin slices
assay, were 0.09 mmoL/mob and 1.74 mmollmob in the glycated J32m was confirmed immunohistochemicabby by using anti-AGE antibody
after a 10-day and 60-day incubation, respectively, and below the and supported by the observation, as described below, that the pen-
detection limit (0.02 mmol/mol) and 2.28 mmol/mol in normal BSA tosidine levels in rat bone particles increased upon incubation with
and the gbycated BSA after a 60-day incubation, respectively. The glucose. Glycated dentin slices obtained in this manner were used as
levels of Amadori product (fructoselysine) were also determined by AGE-modified dentin slices (AGE-dentin slices).
colorimetric assay using a kit (Fructosamine Test Roche-Il; Nihon
Roche Ltd., Tokyo, Japan) (17): 0.05 mob/mob in normal /32m, and Osteoclast-Formation Assay
0.44 mob/mob and 0.70 mob/mob in the glycated f32m after a 10-day The effect of AGE on osteoclast development was examined in two
and 60-day incubation, respectively. different culture systems: the mouse unfractionated bone cell culture
262 Journal of the American Society of Nephrology

B 0 No. of resorption pits I dentin slice

Control

104M

Normal 2M I O-7M
1OM

1O-8M

AGE-fS2M 1O-7M

1O-M *

I cx,25(OH)2D3 I

C No. of TRAP-positive MNCs I dentin slice

0 10 20 40 50
Control

10-EM

Normal 2M 10-7M

10-EM
104M

AGE-2M 10-7M

10M
lcx, 25(OH)2D3 109M *

Figure 1. Effect of normal 2m and AGE-2m purified from the urine of long-term hemodiabysis patients on osteoclast-induced bone
resorption. (A) Microscopy of hematoxylin-stained resorption pits (indicated by arrowheads) on dentin slices cultured for 4 days in the presence
of lO M normal /32m (left panel) or AGE-f32m (right panel). (Original magnification, X40.) (B) The numbers of resorption pits and (C)
TRAP-positive MNC on dentin slices. Mouse unfractionated bone cells (4 X l0 cells/dentin slice) containing TRAP-positive MNC (200
 AGE Enhance Osteoclast-Induced Bone Resorption 263

system and the mouse coculture system of bone marrow cells with Statistical Analyses
primary osteobbast-rich cell populations. For the former system, the Data were expressed as means ± SE from quadrupbicate cultures or
unfractionated bone cell suspension (2 X l0 cells/well) with a five rats. Intergroup differences in means were statistically assessed
TRAP-positive MNC density of 100 cells/well were cultured in a using Dunnett’s multiple-comparison method and by t test.
96-webb plate without dentin slices in the absence of la, 25(OH)2D,
for 4 days. After the depletion of TRAP-positive MNC was con-
firmed, the cells were further incubated for 6 days in the culture
Results
medium containing either l0_6 M AGE-2m or l0_8 M ba,
AGE Enhance Osteoclast-induced Bone Resorption
25(OH)2D,. The number of newly formed TRAP-positive MNC in the
A 4-day culture of unfractionated bone cells containing
culture medium was then counted after TRAP staining of the cells.
Our previous study demonstrated pre-existing TRAP-positive MNC TRAP-positive MNC on dentin slices in the presence of AGE-
are depleted by culturing in the absence of la, 25(OH)2D,, but new 132m purified from the urine of long-term hemodialysis patients
TRAP-positive MNC formation is induced by the addition of la, significantly increased the number of resorption pits formed by
25(OH)2D3 in culture dishes even after the complete depletion of osteoclasts, compared with control cells (Figures 1 , A and B).
TRAP-positive MNC (30). However, there was no statistically significant difference in the
Mouse coculture system of bone marrow cells with osteoblastic number of resorption pits between control cells and those
cells was performed according to the method established by Taka- cultured with normal 2m. An increase of the number of
hashi et al. (31). In brief, bone marrow cells were isolated from tibia resorption pits indicates an acceleration of bone resorption by
of 7- to 9-wk-old mice (32) and primary osteobbastic cells were
osteoclasts. It is notable that AGE-f32m did not affect the
isolated from newborn mice calvaria by collagenase digestion (33).
number of TRAP-positive MNC during the 4-day culture (Fig-
The bone marrow cells (3 X b0 cells/well) and osteoblastic cells (3 X
ure lC), suggesting that pre-existing osteoclasts likely formed
b0 cells/well) were cocultured for 6 days in 0.3 mL of a-MEM
containing 10% FBS with several concentrations of either AGE-2m,
most of the resorption pits. 1 a, 25(OH)2D3, a webb-known
AGE-BSA, or their normal counterparts, lO_8 M la, 25(OH),D,, or bone-resorbing agent that stimulates osteoblasts to release fac-
lO_6 M prostaglandin E2 (Wako Pure Chemicals) in a 48-well plate. tors that affect osteoclasts (25), increased the number of re-
The number of newly formed TRAP-positive MNC was then counted sorption pits together with the increase of the number of
after TRAP staining of the cells. TRAP-positive MNC, in good agreement with our previous
results (30).
In Vivo Resorption Assay The effect of AGE on osteoclast-induced bone resorption
Bone resorption was evaluated in rats that were implanted with was also examined using in vitro preparations of AGE-pro-
devitalized bone particles according to the method of Lian et al. (34). teins. A 4-day culture of the unfractionated bone cells with the
In brief, the femoral bones were removed from I 2- to I 5-wk-obd male glycated 32m after a 60-day incubation (in vitro-prepared
Sprague Dawley rats (Japan Charles River, Tokyo, Japan), stripped of AGE-f32m) increased the number of resorption pits (Figure 2A)
soft tissue, washed in cold ethanol, byophibized, pulverized in a liquid without an increase of the number of TRAP-positive MNC
nitrogen mill, and sieved into 75- to 250-pm particles. These bone
(control, 20.8 ± 3.0 cells/dentin slice; in vitro-prepared AGE-
particles (1 mg) were then incubated with or without 0.5 M glucose in
5.0 mL of 0.3 M phosphate buffer (pH 7.4) at 37#{176}C
for 60 days.
132m, 19.3 ± 2.4 cells/dentin slice). By contrast, there was no
statistically significant difference in the number of resorption
Determination of pentosidine levels by HPLC assay confirmed the
presence of AGE in the glycated bone particles (990 pmollmg), but
pits between control cells and those cultured with the glycated
not in nonglycated counterparts (0.97 pmob/mg). Glycated bone par- 2m after a 10-day incubation, which contained the early
tides obtained in this manner and those obtained by parallel incuba- Amadori products (fructoselysine) but few AGE (assessed by
tion without glucose were used as AGE-modified bone particles its fluorescence [17] and pentosidine level). This indicates that,
(AGE-bone particles) and control bone particles, respectively. among the glycated f32m, the pigmented and fluorescent AGE-
AGE-bone particles or control bone particles (50 mg each) were
132m enhanced bone-resorbing activity of osteoclasts, whereas
packed into l-mL plastic syringes to which 40 L of 10% gelatin was the 2m modified with Amadori products had no such activity.
then added, and were compressed and byophilized. These bone-parti- The bone-resorbing effect of AGE-j32m is thought to be the
cle pellets were then implanted subcutaneously into front chests of
result of not f32m, but of AGE, because in vitro-prepared
five 4-wk-old male Sprague-Dawley rats. Two and 4 wk later, bone
AGE-BSA also increased the number of resorption pits under
particles and surrounding tissues were removed and the bone mineral
the same culture conditions, whereas normal BSA did not
content in these specimens was determined by dual-energy x-ray
absorptiometry using a bone densitometer (Model DSC-600; Aloka,
(Figure 2B).
Tokyo, Japan). All studies were conducted in accord with the National With or without 1 a, 25(OH)2D3, a 4-day culture of unfrac-
Institutes of Health Guide for the Care and Use of Laboratory tionated bone cells on AGE-dentin slices, which were prepared
Animals, and protocols were approved by the institutional Animal by incubating dentin slices with glucose for 40 days, signifi-
Care and Use Subcommittee. cantly increased the number of resorption pits formed by

cells/dentin slice) were cultured on dentin slices for 4 days in a medium containing several concentrations of normal j32m or AGE-f32m, or l0
M liz, 25(OH)2D,. The total number of TRAP-positive MNC on the dentin slices was counted after TRAP staining of the cells. After removal
of the cells, the dentin slices were stained with hematoxylin and the number of densely stained pits on the dentin slices was counted under light
microscopy. Data are expressed as means ± SE from quadruplicate cultures. P < 0.05 versus control by Dunnett’s multiple comparison.
264 Journal of the American Society of Nephrology

A
No. of resorption pits I dentin slice
0

Control

r 1O-7M
Normal 2M I
L 1O-6M
1 1O7M
In vitro-prepared
Amadori-2M [ O-6M

In vitro-prepared 1 1O7M
AGE-2M [ 1O-6M
*

B
No. of resorption pits I dentin slice
0
Control

r 107M
Normal BSA I
L 10-6M
In vitro-prepared [ I 07M
AGE-BSA I
L 106M *

Figure 2. Effect of in vitro-prepared AGE-f32m (A) and AGE-BSA (B) on the number of resorption pits on dentin slices. Mouse unfractionated
bone cells (4 X b0 cells/dentin slice) containing TRAP-positive MNC (200 cells/dentin slice) were cultured on dentin slices for 4 days in a
medium containing several concentrations of glycated proteins after a 10-day (in vitro-prepared Amadori-/32m) or 60-day (in vitro-prepared
AGE-/32m or AGE-BSA) incubation. The number of resorption pits on the dentin slices was then counted. The levels of fructosebysine and
pentosidine in the glycated proteins were discussed in the Methods section. Note that the gbycated proteins after a 60-day incubation contain
both AGE and Amadori products, and that the glycated protein after a 10-day incubation contains Amadori products but few AGE. Data are
expressed as means ± SE from quadruplicate cultures. P < 0.05 versus control by Dunnett’s multiple comparison.

osteocbasts, compared with the cells cultured on control dentin fect on bone resorption. As shown in Figure 4, TRAP-positive
slices upon parallel incubation without glucose (Figure 3). The MNC in the unfractionated bone cells completely disappeared
number of TRAP-positive MNC, however, did not increase by in the culture dishes without dentin slices after a 4-day incu-
being cultured on AGE-dentin slices (20.5 ± 2.6 and 22.8 ± bation in the absence of la, 25(OH)2D3. When la, 25(OH)2D3
2.4 cells/dentin slice in the absence and presence of 10 10 M was added on the fourth day under such culture conditions, the
1 a, 25(OH)2D3, respectively), compared with control dentin number of TRAP-positive MNC increased (open squares),
slices ( 1 9.8 ± 1 .4 and 22.3 ± 2.2 cells/dentin slice in the indicating the formation of new TRAP-positive MNC during
absence and presence of l0 ‘ M la, 25(OH)2D3, respective- the following 6 days of culture. However, both AGE-f32m
by). purified from patients in vivo (open circles) and prepared in
vitro (open triangles) did not increase the number of TRAP-
AGE Have No Effect On New Osteoclast Formation positive MNC. This suggests that AGE have no effect on the
Next, we examined whether AGE had an effect on new formation of new osteoclasts in the mouse unfractionated bone
TRAP-positive MNC formation independently from their ef- cell culture system.
 AGE Enhance Osteoclast-Induced Bone Resorption 265

No. of resorption pits I dentin slice

the action of calcitonin and ipriflavone may be different, be-
0 20 30 cause the latter reduced the number of TRAP-positive MNC

whereas the former did not (Figure 6B). No cytotoxic effects

Control dentin slice -
were observed in calcitonin- or ipriflavone-treated cells on the

AGEdentin slice - 11* dentin

blue
slices
staining.
after a 4-day incubation, when assessed by trypan

AGE Enhance Bone Resorption in Rats implanted with

Control dentin slice +
1 Bone Particles
k The pathophysiobogicab significance of AGE in bone resorp-
AGE-dentin slice + ] tion hinges upon the in vivo demonstration of enhanced bone
resorption by AGE modification. Therefore, AGE-bone parti-
des or control bone particles were implanted subcutaneously
la, 25(OH)D3
(101OM) into rats and their resorption was examined. Histological ex-
amination revealed the presence of MNC in resorptive bacunae
Figure 3. Effect of AGE modification of dentin slices on the number
of bone particles (data not shown). Bone particles were
of resorption pits. Mouse unfractionated bone cells (4 X l0 cells/
resorbed with time, and 60.8% of AGE-bone particles and
dentin slice) containing TRAP-positive MNC (200 cells/dentin slice)
were cultured in the presence or absence of 10 M
‘#{176} 1a, 25(OH),D3 43.7% of control bone particles were resorbed after 4 wk of
for 4 days on dentin slices, which had been incubated with glucose implantation (Figure 7). Notably, the degree of resorption of
(AGE-dentin slices) or without glucose (control dentin slices) for 40 AGE-bone particles was much greater than that of control bone
days. The number of resorption pits on the dentin slices was counted. particles: the amount of the remaining AGE-bone particles
Data are expressed as means ± SE for quadruplicate cultures. *p < after 4 wk was 69.8% of that of the control group.
0.05 versus control dentin slice by t test.
Discussion
The study presented here has provided in vitro and in vivo
Osteoblastic cells are required for the differentiation of evidence that AGE enhance osteoclast-induced bone resorp-
osteoclast progenitors into osteoclasts and the efficiency of tion. First, when mouse unfractionated bone cells containing
osteoclast formation is greatly enhanced by the presence of osteoclasts were cultured on dentin slices, AGE-2m, but not
osteoblastic cells (25,3 1-33). Takahashi et al. (3 1) demon- normal f3,m, purified from the urine of bong-term hemodialysis
strated that various colony-stimulating factors stimulated Os- patients increased the number of resorption pits formed by
teoclast formation in the coculture system of bone marrow cells osteoclasts. Second, AGE-/32m and AGE-BSA prepared in
with osteobbastic cells, whereas these factors did not stimulate vitro by incubating with glucose for 60 days increased the
in the bone marrow culture system alone, thereby suggesting number of resorption pits, whereas their normal counterparts or
that the coculture system is suitable for examining the effect on those modified with the early glycation products did not. Third,
osteoclast formation. We therefore used a cocubture system of the osteoclast-induced bone resorption was enhanced on AGE-
bone marrow cells with primary osteoblastic cells isolated from dentin slices, compared with unmodified dentin slices. Fourth,
mouse calvaria. However, even in the presence of osteobbastic the effect of AGE on bone resorption was effectively inhibited
cells, no TRAP-positive MNC were formed after adding AGE- by calcitonin and ipriflavone, both of which are inhibitors of
132m or AGE-BSA into the culture dishes (Figure 5). By bone resorption. The results from these in vitro studies were
contrast, many TRAP-positive MNC appeared in response to further confirmed by in vivo evidence that bone particles upon
la, 25(OH)2D3 or prostaglandin E2. incubation with glucose for 60 days (AGE-bone particles),
when implanted subcutaneously in rats, were resorbed to a
Calcitonin and Ipriflavone Effectively Inhibit AGE- much greater extent than control bone particles upon parallel
Enhanced Bone Resorption incubation without glucose. These findings suggest that the
Both calcitonin and ipriflavone are known to prevent bone modification of bone matrix proteins with AGE might, in part,
loss in various types of animal models with experimentally play a robe in the remodeling of senescent bone matrix tissues.
induced osteoporosis, and are also known to be effective for The AGE-enhanced bone resorption is also supported by our
the treatment of osteoporosis in humans (35-37). We examined preliminary data that AGE-f31m, whether purified from pa-
the effect of calcitonin and ipriflavone on AGE-enhanced bone tients in vivo or prepared in vitro, induces net calcium efflux
resorption. Unfractionated bone cells containing TRAP-posi- from cultured neonatal mouse calvaria to a much greater extent
tive MNC were cultured for 4 days in the medium containing than normal f32m (Sprague 5, and Miyata T, unpublished
in vitro-prepared AGE-32m in the presence ofcabcitonin (lO_8 observation).
M) or ipriflavone (l0 M). A previous study demonstrated The finding that AGE did not increase the number of newly
that both agents, at these concentrations, significantly reduced formed osteoclasts in the coculture system of bone marrow
the number of resorption pits induced by la, 25(OH)2D3 (30). cells with osteobbastic cells, as well as in the unfractionated
Both calcitonin and ipriflavone significantly (P < 0.05) re- bone cell culture system, likely suggests that AGE have no
duced the number of resorption pits induced by in vitro- effect on osteoclast formation. We previously demonstrated
prepared AGE-f32m (Figure 6A). However, the mechanisms of that AGE-32m, but not normal 32m, stimulates human mono-
266 Journal of the American Society of Nephrobogy

100

0)
80
(I)
C-)
z
0) 60
>
Cl)
0
d.
I-.
I.-

0
0
z

2 4 6 8
Days
Figure 4. Effect of AGE-32m on new TRAP-positive MNC formation. Ike-existing TRAP-positive MNC in mouse unfractionated bone cells
(2 X b0 cells/well) were depleted by being cultured in a 96-webb plate for 4 days in the absence of 1a, 25(OH)2D3 (closed circles). On the
fourth day (arrow), l06 M either AGE-32m purified from the urine of long-term hemodialysis patients (open circles) or in vitro-prepared
AGE-32m (open triangles), or l0_8 M ba, 25(OH)2D3 (open squares) was supplemented into the culture medium, and the cells were further
incubated for 6 days without dentin slices. The number of newly formed TRAP-positive MNC was then counted after TRAP staining. Data are
expressed as means ± SE from quadrupbicate cultures.

nuclear phagocytes to secrete cytokines such as IL-1f3 (7,17). alternative possibility is that osteoclasts are stimulated by
Although IL-b 3 is a potent stimulator of osteoclast formation, soluble factor(s) that are released from osteoblasts by AGE, as
the concentration of IL-l3 necessary for this process is ap- demonstrated previously as the mechanism of stimulation of
proximately 1 ng/m.L (38), which is much higher than that osteoclasts by parathyroid hormone (39,40) and la,
secreted from human mononuclear phagocytes by the action of 25(OH)2D3 (41). Second, the modification of bone matrix
AGE-J32m (7, 1 7). However, although concentrations of Se- proteins with AGE might create a microenvironment favorable
creted IL-l may not have been sufficient to stimulate osteo- for bone resorption and facilitate osteoclasts to resorb bone
clast formation in the cell culture system, AGE-induced secre- matrices, e.g. , by strengthening the attachment of osteoclasts to
tion of IL-lf3 in vivo may yield high local concentrations of AGE-modified bone surface or by making AGE-modified bone
IL-i f3, sufficient to act in a paracrine manner to induce differ- matrices susceptible to degradation with bysosomal enzymes
entiation of osteoclast progenitors in tissues. At least, the secreted by osteoclasts. A previous study demonstrated the
mechanism by which AGE enhance bone resorption in the alteration in the integrity of articular cartilage as a result of the
present cell culture system is thought not to promote differen- crosslinks of pentosidine (42), thereby implicating possible
tiation of osteoclast progenitors into osteoclasts. involvement in matrix degradation of osteoarthritis. Further
At present, this mechanism remains unknown. However, study will undoubtedly be necessary to elucidate the molecular
two possible explanations may be considered. First, AGE mechanism of AGE-enhanced bone resorption by osteoclasts.
might stimulate the activity of osteoclasts. It is inconclusive Dialysis-rebated amyloidosis is an osteoarticular complica-
whether or not AGE can activate osteoclasts directly. An lion of long-term hemodialysis patients (21,22). Subchondral
 AGE Enhance Osteoclast-Induced Bone Resorption 267

No. of TRAP-positive MNCs I well

0 200 400 600 800 1000 1200
I I i I i I i I i I i I

Control
-107M
Normal 2M Llo..6M
107M
AGE-2M L10-6M

In vitro-prepared 1 07M

AGE-2M L10-6M
107M
Normal BSA L10-6M

In vitro-prepared 1 0-7M
AGE-BSA L10-6M
1 a, 25(OH)2D3 1 0-8M *

Prostaglandin E2 10-6M -1*

Figure 5. Effect of AGE-modification on new TRAP-positive MNC formation in the coculture system of bone marrow cells and osteobbastic
cells. Mouse bone marrow cells (3 X l0 cells/webb) were cocultured with primary osteobbastic cells (3 X l0 cells/webb) isolated from calvaria
for 6 days in 48-well plate in the presence of several concentrations of either AGE-f32m purified from patients in vivo, in vitro-prepared
AGE-f32m or AGE-BSA, or their normal counterparts, lO_8 M Ia, 25(OH)2D3, or b06 M prostaglandin E2. The number of newly formed
TRAP-positive MNC was then counted after TRAP staining. Data are expressed as means ± SE from quadruplicate cultures. P < 0.01 versus
control by Dunnett’s multiple comparison.

erosion and bone cysts as a result of an accelerated bone patients and elderly subjects, compared with healthy young
resorption and replacement of bone by amyloid deposits are subjects (23,24), the study presented here suggests a potential
becoming a serious problem, and are causing pathological link of the accumulation of AGE in bone matrix tissues to
fractures (21,22,43). Although 2m is shown to be a major osteoporosis associated with diabetes and aging. However,
constituent in amyboid fibrils (44,45), the mechanism of bone because bone resorption is a combined result that is determined
resorption has remained unknown. We previously demon- by many hormonal and local factors, more study will be
strated that AGE are present in f32m-forming amyloid fibribs of necessary to clarify the issue as to how much the AGE mod-
dialysis-related amyboidosis (14,15). The results of the study ification is clinically relevant to bone resorption in these pa-
presented here suggest that AGE-2m in amyloid fibrils accel- tients.
erates bone resorption around long-lived amyboid deposits, Finally, this study’s observations that both calcitonin and
implicating a role of AGE in the pathogenesis of dialysis- ipriflavone effectively inhibit AGE-enhanced bone resorption,
related amyloidosis, especially in bone resorption. with the concentrations roughly corresponding to plasma con-
Our preliminary immunohistochemical studies using anti- centrations when administered in vivo at therapeutic doses
AGE and anti-pentosidine antibodies revealed that human bone (47), suggest the possible effectiveness of these agents on
tissues obtained from elderly subjects contain a significant AGE-enhanced bone resorption, such as dialysis-related amy-
amount of AGE, compared with those from young subjects loidosis, for which there is no effective method available for
(Miyata T, and Kawai R, unpublished observation). This is not prevention and treatment.
surprising, because the AGE modification occurs in long-lived
matrix proteins such as Type I collagen, a major component of Acknowledgments
bone matrix (46). Taking into account the observations that the The authors give special thanks to Drs. Naoyuki Takahashi and
AGE levels in matrix tissues are markedly elevated in diabetic Tatsuo Suda, Department of Biochemistry, Showa University School
268 Journal of the American Society of Nephrology

A No. of resorption pits I dentin slice

0 10 20

Control [
L+

Calcitonin [
(10-8M) [+ §4:1:

Ipriflavone [
(10-5M) [+ 1

In vitro-prepared
AG E-2M
(10-6 M)

B No. of TRAP-positive MNCs I dentin slice

0 10 20 30

Control [
L+

Calcitonin [-
(10-8M) [+

Ipriflavone [
(10-5M) [
In vitro-prepared
AGE-2M
(10-6 M)
Figure 6. Inhibitory effect of calcitonin and ipriflavone on AGE-enhanced bone resorption. Mouse unfractionated bone cells (4 X l0
cells/dentin slice) containing TRAP-positive MNC (200 cells/dentin slice) were cultured for 4 days in the medium with or without lO_6 M in
vitro-prepared AGE-f32m in the presence or absence of l0_8 M chick calcitonin or l0 M ipriflavone. The numbers of resorption pits (A)
and TRAP-positive MNC (B) on the dentin slices were then counted. Data are expressed as means ± SE from quadruplicate cultures. *p <
0.05 and tp < 001 control without in vitro-prepared AGE-f32m in the absence of calcitonin and ipriflavone, P < 0.05 and ‘P < 0.01 versus
control with in vitro-prepared AGE-2m in the absence of calcitonin and ipriflavone, and #p < 0.05 and tp < 0.01 versus the sample without
in vitro-prepared AGE-/32m, by t test.
 AGE Enhance Osteoclast-tnduced Bone Resorption 269

of hemodialysis-associated amyboidosis. Biochem Biophys Res

Bone mineral content (mg)
In fl in 40 Commun 201: 1235-1241, 1994
Initial bone particles 9. Neeper M, Schmidt AM, Brett J, Yan SD, Wang F, Pan YC,
Ebbiston K, Stern D, Shaw A: Cloning and expression of a cell
Control bone particles (2 weeks) surface receptor for advanced glycosylation end products of
LH
proteins. J Biol Chem 267: 14998-15004, 1992
AGE-bone particles (2 weeks)
10. Schmidt AM, Yan SD, Brett J, Mora R, Nowygrod R, Stern D:
Regulation of human mononuclear phagocyte migration by cell
Control bone particles (4 weeks)
surface-binding proteins for advanced glycation end products.
AGE.bone particles (4 weeks) J Clin Invest 92: 2155-2168, 1993
*
1 1. Baynes JW: Perspectives in diabetes: Robe of oxidative stress in
(Time after Implantation)
development of complications in diabetes. Diabetes 40: 405-
Figure 7. Effect of AGE modification on bone resorption in rats 412, 1991
implanted with bone particles. Bone particles were prepared from rat 12. Yan SD, Schmidt AM, Anderson GM, Zhang J, Brett J, Zou YS,
femoral bones, incubated with glucose (AGE-bone particles) or with- Pinsky D, Stern D: Enhanced cellular oxidant stress by the
out glucose (control bone particles) for 60 days, and implanted sub- interaction of advanced glycation end products with their recep-
cutaneously into five rats. Two and 4 wk later, bone particles and tors/binding proteins. J Biol Chem 269: 9889-9897, 1994
surrounding tissues were removed and the bone mineral content was 13. Bucala R, Makita Z, Vega G, Grundy 5, Koschinsky T, Cerami
determined by dual energy x-ray
absorptiometry. Data are expressed A, Vlassara H: Modification of low density bipoprotein by ad-
as means ± SE from five rats. *p < 0.01 versus control bone particles vanced glycation end products contributes to the dyslipidemia of
by t test. diabetes and renal insufficiency. Proc Nat! Acad Sci USA 91:
9441-9445, 1994
14. Miyata T, Oda 0, Inagi R, lida Y, Araki N, Yamada N, Horiuchi
5, Taniguchi N, Maeda K, Kinoshita T: 32-Microgbobulin mod-
of Dentistry, for their excellent technical expertise and invaluable ified with advanced glycation end products is a major component
comments. We also thank Dr. Hisao Seo for helpful discussion. This of hemodialysis-associated amyloidosis. J Clin Invest 92: 1243-
study was supported by grants from the Research for the Future 1252, 1993
Program of the Japan Society for the Promotion of Science 15. Miyata T, Taneda 5, Kawai R, Ueda Y, Horiuchi 5, Hara M,
(96L00303) and from the Baxter Extramural Grant Program. Maeda K, Monnier VM: Identification of pentosidine as a native
structure for advanced glycation end products in j32-microgbobu-

References bin forming amyloid fibrils in patients with dialysis-rebated amy-

1. Brownbee M, Cerami A, Vbassara H: Advanced glycosybation boidosis. Proc Nat! Acad Sci USA 93: 2353-2358, 1996
end products in tissue and the biochemical basis of diabetic 16. Miyata T, Inagi R, Wada Y, Ueda Y, Iida Y, Takahashi M,
complications. N Engl J Med 318: 1315-1321, 1988 Taniguchi N, Maeda K: Glycation of human /32-microglobubin in
2. Baynes JW, Monnier VM: The Maiblard reaction in aging, dia- patients with hemodiabysis-associated amyboidosis: Identification
betes and nutrition. Prog Clin Biol Res 304: 1-410, 1989 of the glycated sites. Biochemistry 33: 122 15-1 2221 , I 994
3. Kirstein M, Brett J, Radoff 5, Ogawa 5, Stem D, Vlassara H: 17. Miyata T, Iida Y, Ueda Y, Shinzato T, Seo H, Monnier VM,
Advanced protein gbycosylation induces transendothelial human Maeda K: Monocyte/macrophage response to 32-microgbobubin
monocyte chemotaxis and secretion of platelet-derived growth modified with the advanced glycation end products. Kidney mt
factor: Role in vascular disease of diabetes and aging. Proc Nail 49: 538-550, 1996
Acad Sci USA 87: 9010-9014, 1990 18. Smith MA, Taneda S, Richey PL, Miyata 5, Yan SD, Stem D,
4. Vlassara H, Brownbee M, Manogue KR, Dinarello CA, Pasagian Sayre LM, Monnier VM, Perry G: Advanced Maillard reaction
A: CachectinfTNF and IL-l induced by glucose-modified pro- end products are associated with Alzheimer disease pathology.
teins: role in normal tissue remodeling. Science 240: 1546-1548, Proc Nail Acad Sci USA 91 : 57 10-57 14, 1994
1988 19. Vitek MP, Bhattacharya K, Gbendening JM, Stopa E, Vbassara H,
5. Skolnik EY, Yang Z, Makita Z, Radoff 5, Kirstein M, Vlassara Bucala R, Manogue K, Cerami A: Advanced gbycation end
H: Human and rat mesangial cell receptors for glucose-modified products contribute to amyloidosis in Abzheimer disease. Proc
proteins: Potential role in kidney tissue remodelling and diabetic Nat! Acad Sci USA 9 1: 4766-4770, 1994
nephropathy. J Exp Med 174: 931-939, 1991 20. Yan SD, Yan FF, Chen X, Jin F, Chen M, Kuppusamy P, Smith
6. Doi T, Vlassara H, Kirstein M, Yamada Y, Striker GE, Striker MA, Perry G, Godman GC, Nawroth P. Zweier JL, Stern D:
U: Receptor-specific increase in extracelbular matrix production Non-enzymaticably gbycated tau in Alzheimer’ s disease induces
in mouse mesangiab cells by advanced glycosylation end prod- neuronal oxidant stress resulting in cytokine gene expression and
ucts is mediated via platelet-derived growth factor. Proc Nail release of amyboid j3-peptide. Nature Med 1: 693-699, 1995
Acad Sci USA 89: 2873-2877, 1992 21. DrUeke TB: Beta-2-microgbobubin amyboidosis and renal bone
7. Miyata T, Inagi R, Iida Y, Sato M, Yamada N, Osa 0, Maeda K, disease. Miner Electrolyte Metab 17: 261-272, 1991
Seo H: Involvement of f32-microgbobulin modified with ad- 22. Koch KM: Dialysis-related amyboidosis. Kidney Im’ 41 : 1416-
vanced glycation end products in the pathogenesis of hemodial- 1429, 1992
ysis-associated amyboidosis: Induction of human monocyte che- 23. Makita Z, Radoff 5, Rayfield EJ, Yang Z, Skolnik E, Delaney V.
motaxis and macrophage secretion of tumor necrosis factor-a Friedman EA, Cerami A, Vlassara H: Advanced glycosylation
and interleukin 1. J Clin Invest 93: 521-528, 1994 end products in patients with diabetic nephropathy. N Eng!J Med
8. lida Y, Miyata T, Inagi R, Sugiyama 5, Maeda K: p2-Micro- 325: 836-842, 1990
globulin modified with advanced glycation end products induces 24. Dyer DG, Dunn JA, Thorpe SR. Bailie KE, Lyons TJ, McCance
interleukin-6 from human macrophages: Role in the pathogenesis DR, Baynes JW: Accumulation of Maillard reaction products in
270 Journal of the American Society of Nephrology

skin collagen in diabetes and aging. J Clin mnvest 91 : 2463-2469, 37. Reginster JY: Ipriflavone pharmacological properties and usefulness
I993 in postmenopausab osteoporosis. Bone Mm 23: 223-232, 1993
25. Suda T, Takahashi N, Martin TJ: Modulation of osteoclast dif- 38. Akatsu T, Takahashi N, Udagawa N, Imamura K, Yamaguchi A,
ferentiation. Endocrine Rev 13: 66-80, 1992 Sato K, Nagata N, Suda T: Robe of prostaglandins in interleukin-
26. Sell DR. Monnier VM: Structure elucidation of a senescence b-induced bone resorption in mice in vitro. J Bone Miner Res 6:
cross-link from human extracellubar matrix. J Biol Chem 264: 183-189, 1991
21597-21602, 1989 39. McSheehy PMJ, Chambers TJ: Osteoblastic cells mediate oste-
27. Odetti P. Forgarty J, Sell DR. Monnier VM: Chromatographic oclastic responsiveness to parathyroid hormone. Endocrinology
quantitation of plasma and erythrocyte pentosidine in diabetic 118: 824-828, 1986
and uremic subjects. Diabetes 41: 153-159, 1992 40. McSheehy PMJ, Chambers TJ: Osteoblast-like cells in the pres-
28. Takada Y, Kusuda M, Hiura K, Sato T, Mochizuki H, Nagao Y, ence of parathyroid hormone release soluble factor that stimu-
Tomura M, Yashiro M, Hakeda Y, Kawashima H, Kumegawa
bates osteoclastic bone resorption. Endocrinology 1 19: 1654-
M: A simple method to assess osteoclast-mediated bone resorp-
1659, 1986
tion using unfractionated bone cells. Bone Mm 17: 347-359,
41. McSheehy PMJ, Chambers TJ: 1, 25-Dihydroxyvitamin D3 stim-
I992
ubates rat osteobbastic cells to release a soluble factor that in-
29. Burstone MS: Histochemical demonstration of acid phosphatase
creases osteoclastic bone resorption. J Clin Invest 80: 425-429,
with naphthol AS-phosphate. J Nat! Cancer inst 21 : 523-539,
1987
1958
42. Pokharna HK, Monnier VM, Boja B, Moskowitz RW: Lysyl
30. Notoya K, Yoshida K, Taketomi 5, Yamazaki I, Kumegawa M:
oxidase and Maiblard reaction-mediated crosslinks in aging and
Inhibitory effect of ipriflavone on osteoclast-mediated bone re-
osteoarthritic rabbit cartilage. J Orthop Res 13: 13-21, 1995
sorption and new osteoclast formation in bong-term cultures of
43. van Ypersebe de Strihou C, Honhon B, Vandenbroucke JM,
mouse unfractionated bone cells. Calcif Tissue mt 53: 206-209,
Huaux JP, Noel H, Maldague B: Dialysis amyboidosis. Adv
I 993
Nephrol 17: 401-422, 1988
3 1. Takahashi N, Udagawa N, Akatsu T, Tanaka H, Shionome M,
44. Gejyo F, Yamada T, Odani 5, Nakagawa Y, Arakawa M, Kuni-
Suda T: Role of colony-stimulating factors in osteoclast devel-
tonmo T, Kataoka H, Suzuki M, Hirasawa Y, Shirahama T,
opment. J Bone Miner Res 6: 977-985, 1991
32. Takahashi N, Yamana H, Yoshiki 5, Roodman DG, Mundy GR, Cohen AS, Schmid K: A new form of amyboid protein associated
with chronic hemodialysis was identified as f32-microgbobulin.
Jones Si, Boyde A, Suda T: Osteoclast-like cell formation and its
regulation by osteotropic hormones in mouse bone marrow cub- Biochem Biophys Res Commun 129: 701-706, 1985

tures. Endocrinology 122: 1373-1382, 1988 45. Gorevic PD, Munoz PC, Casey TT, DiRaimondo CR, Stone WJ,
33. Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A, Prebli FC, Rodrigues MM, Poulik MD, Frangione B: Polymer-
Moseley JM, Martin Ti, Suda T: Osteobbastic cells are involved ization of intact f32-microgbobulin in tissue causes amyboidosis in
in osteoclast formation. Endocrinology 123: 2600-2602, 1988 patients on chronic hemodialysis. Proc Nat! Acad Sci USA 83:
34. Lian lB. Tassinari M, Glowacki J: Resorption of implanted bone 7908-7912, 1986
prepared from normal and warfarin-treated rats. J Clin mnvest 73: 46. Kahn AJ, Fabbon MD, Teitelbaum SL: Structure-function rela-
1223-1226, 1984 tionships in bone: An examination of events at the cellular bevel.
35. Wronski TJ, Yen C-F, Burton KW, Mehta RC, Newman PS, In: Bone and Mineral Research, edited by Peck WA, Amster-
Sobtis EE, Deluca PP: Skeletal effects of calcitonin in ovariec- dam, Ebsevier, 1984, pp 125-174
tomized rats. Endocrinology 129: 2246-2250, 1991 47. Yoshida K, Tsukamoto T, Tanayama 5, Uemura I, Shibata K:
36. Reginster JY: Effect of cabcitonin on bone mass and fracture Disposition of ipriflavone (TC-80) in rats and dogs. Radioiso-
rates. Am J Med 91[Suppl SB]: 195-225, 1991 topes 34: 618-623, 1985