173
Estrogen receptor beta is a therapeutic target to improve
osteoporotic cortical bone repair in mice: An in-vivo longitudinal
study by high-resolution microCT
Yixin He
1
, Ge Zhang
2
, Zhong Liu
2
, Xiaohua Pan
2
, Xinhui Xie
2
,
Xinluan Wang
2
, Lizhen Zheng
2
, Ling Qin
2
1
The Chinese University of Hong Kong, HongKong SAR, China
2
Department of Orthopaedics & Traumatology, The Chinese University Of
Hong Kong, Hong Kong SAR, China
Introduction: Orthopaedic surgeons are challenged by impaired
or delayed fracture repair in osteoporotic bone. Therapeutic strata-
gem should be defined according to identified molecular target.
However, the molecular mechanism of osteoporotic fracture repair
remains poorly understood. Intramembranous ossification (radial
bone growth) and endochondral ossification (linear bone growth)
are two important processes in callus hardening during fracture
repair. Estrogen-receptor-alpha (ERalpha), mediating the classical
estrogen signaling pathway, exerts a positive effect on bone
maintenance, but the role of ERalpha in regulation of bone
d eve l op m en t rem a in s c on trove r si a l [ 1, 2 ] . P rev io u s re s ea rc h es d e-
monstrate estrogen depletion induces a significant increase in
Estrogen-receptor-beta (ERbeta) expression in bone. ERbeta signal-
ing participates in inhibiting both intramembranous ossification and
e n d oc h on d ra l os s if i c a t io n [ 1, 3 ] , T h e ref o re, we f o rm t h e hyp ot h es i s
that blockade of ERbeta could promote osteoporotic fracture repair.
Methods: Thirty ERbeta knockout (KO) and 30 wild-type female
mice (WT, C57BL/6) aged 3 months were used in this study. All mice
were ovariectomized first. 6 weeks after ovariectomy, bilateral
0.8 mm-diamter drill holes were made from the posterior to the
anterior of the diaphysis of the femur. High-resolution Micro-CT
(VivaCT 40, Scanco) was employed to in vivo monitoring the repair
process at day 0, 3, 7, 10, 14 and 21. The bone volume fraction (BV/TV)
and bone mineral density (BMD) were evaluated at both defect site
and intra-medulla space in two groups. Repeat measure ANOVA and
Independent t-test were performed to analyze the data. Results &
discussion: With regard to Micro-CT measurement, WT and KO mice
differed significantly in the pattern of the change in the BMD over
time in both defect region and intra-medulla space (p < 0.05 for the
interaction between time and group by the repeat measure ANOVA).
In the defect region, the BMD increased to a similar extent from
baseline in both WT and KO mice at 3 days post-fracture. At day 7, a
significant increase in the BMD over baseline was apparent in both
WT and KO mice, but the extent of this increase differed between the
2 groups (+45% for WT and +78% for KO; p<0.05versusbaselinefor
both). Thereafter, a continuous increase in BMD to the peak at day 14
and followed by a decline at day 21 was apparent in both groups. The
BMD was significantly higher in KO mice compared with WT mice
from day 7 to day 21. In the intra-medulla space, the change of BMD
within defect region showed similar pattern with defect region in
both groups from day 0 to day 14, except the BMD at day 21 returns to
the baseline level in both groups. In addition, the BV/TV data was
consistent with the findings from BMD measurement. A higher BMD
within defect region in KO group compared to WT group suggested
that osteogenesis was promoted by blockade of ERbeta pathway
during osteoporotic bone healing. In both groups, callus remodeling
was present after 14 days post-fracture. The returning of BMD to the
baseline level within intra-medulla space in both groups at 21 days
post-fracture suggested that the blockade of ERbeta pathway didn't
affect remodeling, which was not like delayed remodeling by PTH
t rea t m e n t [ 4 ] , a le n d ron a te tre a t m e n t [ 5 ] o r z ol ed ro n ic a c i d t rea t -
m e n t [ 6 ] . T h e re s ul t s fu r th er s u gge s te d bl oc k a d e of E Rb e t a c o ul d
promote osteoporotic fracture repair. Conclusion: This is the first
study employing high-resolution Micro-CT to in vivo monitoring the
fracture repair. ERbeta pathway is a potential therapeutic target for
promoting osteoporotic fracture repair. Highly selective ERbeta
antagonist should be investigated as an osteoporotic fracture repair
drug.
Acknowledgment
This study was supported by AO Research Grant S-08-74Z.
References
[1] Chagin AS, et al. Estrogen receptor-beta inhibits skeletal growth and has the capacity
to mediate growth plate fusion in female mice. J Bone Miner Res 2004;19:72–7.
[2] Tozum TF, et al. Effects of sex steroid receptor specificity in the regulation of skeletal
metabolism, Calcif. Tissue Int 2004;75:60–70.
[3] Ke HZ, et al. The role of estrogen receptor-beta, in the early age-related bone gain and
later age-related bone loss in female mice. J Musculoskelet Neuronal Interact
2002;2:479–88.
[4] Komatsu DE, et al. Longitudinal in vivo analysis of the region-specific efficacy of
parathyroid hormone in a rat cortical defect model. Endocrinology 2009;150:1570–9.
[5] Odvina CV, et al. Severely suppressed bone turnover: a potential complication of
alendronate therapy. J Clin Endocrinol Metab 2005;90:1294–301.
[6] McDonald MM, et al. Bolus or weekly zoledronic acid administration does not delay
endochondral fracture repair but weekly dosing enhances delays in hard callus
remodeling. Bone 2008;43:653–62.
doi:10.1016/j.bone.2010.09.196
176
Research on relationship between FPN1 content and effect
of hepcidin on intracellular calcium and iron ions change
in osteoblasts
Yong Ma, You-jia Xu
Department of Orthopaedics, The Second Affiliated Hospital of Soochow
University, Suzhou, Jiangsu, China
Objective: Iron metabolism and bone metabolism have been
found to gradually have a close relationship. Hepcidin is a central
regulation peptide of iron metabolism in vivo and FP1 is the binding
target molecule of hepcidin to play an iron metabolism regulation
role. In this research, we investigate the relationship between FPN1
expression and effect of hepcidin on intracellular calcium and iron
ions change and make a preliminary mechanism analysis of hepcidin
on osteoblasts. Methods: hFOB 1.19 cells were treated with different
concentrations of hepcidin. A confocal laser scanning microscope
(CLSM) was used to observe intracellular calcium and iron fluores-
cence intensity. We detected FPN1 protein content by Western blot.
Results: In test concentration, the fluorescence intensity of intracel-
lular iron gradually weakened with hepcidin as concentration
increased suggesting that the intracellular iron concentration
increased. Meanwhile, calcium fluorescence intensity gradually
increased with extracellular hepcidin concentration increase. Fluor-
escence intensity of intracellular calcium increase suggested that
intracellular calcium increased. FPN1 protein content was depressed
in the present of hepcidin. There was a negative correlation between
FP1 content and hepcidin concentration. Conclusions: Hepcidin
administration increased calcium and iron in osteoblasts, which
indicated that hepcidin might prompt calcium and iron influx
transport to affect the function of osteoblasts. We also found that
the FPN1 protein is present in osteoblasts, which is the target
molecule of hepcidin. FPN1 content had a negative correlation with
hepcidin concentration. We considered that hepcidin might affect
osteoblasts through the pathway of FPN1 content regulation. The
application of hepcidin might influence iron metabolism related bone
metabolism and effects on hepcidin might be a new direction for
further bone metabolism studies.
doi:10.1016/j.bone.2010.09.197
Abstracts / Bone 47 (2010) S385–S458S406