Systemic transplantation of human adipose‐derived stem cells stimulates bone repair by promoting osteoblast and osteoclast function

Article (PDF Available)inJournal of Cellular and Molecular Medicine 15(10):2082-94 · December 2010with24 Reads
DOI: 10.1111/j.1582-4934.2010.01230.x · Source: PubMed
Abstract
Systemic transplantation of adipose-derived stem cells (ASCs) is emerging as a novel therapeutic option for functional recovery of diverse damaged tissues. This study investigated the effects of systemic transplantation of human ASCs (hASCs) on bone repair. We found that hASCs secrete various bone cell-activating factors, including hepatocyte growth factor and extracellular matrix proteins. Systemic transplantation of hASCs into ovariectomized mice induced an increased number of both osteoblasts and osteoclasts in bone tissue and thereby prevented bone loss. We also observed that conditioned medium from hASCs is capable of stimulating proliferation and differentiation of osteoblasts via Smad/extracellular signal-regulated kinase (ERK)/JNK (c-jun NH(2) -terminal kinase) activation as well as survival and differentiation of osteoclasts via ERK/JNK/p38 activation in vitro. Overall, our findings suggest that paracrine factors secreted from hASCs improve bone repair and that hASCs can be a valuable tool for use in osteoporosis therapy.
Systemic transplantation of human adipose-derived stem cells
stimulates bone repair by promoting osteoblast
and osteoclast function
Kyunghee Lee
a, b, #
, Hyunsoo Kim
a, b, #
, Jin-Man Kim
a, b
, Jae-Ryong Kim
b, c
, Keuk-Jun Kim
d
,
Yong-Jin Kim
e
, Se-Il Park
f
, Jae-Ho Jeong
g
, Young-mi Moon
g
, Hyun-Sook Lim
h
, Dong-Won Bae
i
,
Joseph Kwon
j
, Chang-Yong Ko
k
, Han-Sung Kim
k
, Hong-In Shin
l
, Daewon Jeong
a, b,
*
a
Department of Microbiology, Yeungnam University College of Medicine, Daegu, Korea
b
Aging-associated Vascular Disease Research Center, Yeungnam University College of Medicine, Daegu, Korea
c
Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu, Korea
d
Department of Clinical Pathology, Taekyeung College, Gyeungsan, Korea
e
Department of Pathology, Yeungnam University College of Medicine, Daegu, Korea
f
Department of Orthopedic Surgery, Yeungnam University College of Medicine, Daegu, Korea
g
Noblesse Plastic Surgery & Stem Tec Korea, Daegu, Korea
h
Department of Public Health Administration, Hanyang Women’s University, Seoul, Korea
i
Central Instrument Facility, Gyeongsang National University, Jinju, Korea
j
Gwangju Center, Korea Basic Science Institute, Gwangju, Korea
k
Department of Biomedical Engineering, College of Health Science, Institute of Medical Engineering, Yonsei University, Wonju, Korea
l
IHBR, Department of Oral Pathology, School of Dentistry, Kyungpook National University, Daegu, Korea
Received: August 20, 2010; Accepted: November 2, 2010
Abstract
Systemic transplantation of adipose-derived stem cells (ASCs) is emerging as a novel therapeutic option for functional recovery of
diverse damaged tissues. This study investigated the effects of systemic transplantation of human ASCs (hASCs) on bone repair. We
found that hASCs secrete various bone cell-activating factors, including hepatocyte growth factor and extracellular matrix proteins.
Systemic transplantation of hASCs into ovariectomized mice induced an increased number of both osteoblasts and osteoclasts in bone
tissue and thereby prevented bone loss. We also observed that conditioned medium from hASCs is capable of stimulating proliferation
and differentiation of osteoblasts
via
Smad/extracellular signal-regulated kinase (ERK)/JNK (c-jun NH
2
-terminal kinase) activation as well
as survival and differentiation of osteoclasts
via
ERK/JNK/p38 activation
in vitro
. Overall, our findings suggest that paracrine factors
secreted from hASCs improve bone repair and that hASCs can be a valuable tool for use in osteoporosis therapy.
Keywords: adipose-derived stem cell
osteoblast
osteoclast
osteoporosis
systemic transplantation
J. Cell. Mol. Med. Vol 15, No 10, 2011 pp. 2082-2094
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
doi:10.1111/j.1582-4934.2010.01230.x
Introduction
Osteoporosis is a prevalent bone disease that is characterized by
loss of bone mass and strength, leading to fragility fracture [1].
Osteoblasts, which are derived from mesenchymal stem cells
(MSCs), are ultimately responsible for bone formation; osteo-
clasts are derived from pluripotent haematopoietic cells and are
capable of resorbing bone. During adult life, bone is continuously
remodelled by orchestrated cross-talk between osteoblasts and
osteoclasts [2], and an imbalance in their function results in
decreased bone quality, most commonly represented by the
osteoporotic phenotype. A relatively higher bone resorption
activity by osteoclasts than bone formation activity by osteoblasts
leads to bone loss. High- and low-turnover osteoporotic
#
These authors contributed equally to this work.
*Correspondence to: Daewon JEONG,
Department of Microbiology, Yeungnam University
College of Medicine, Daegu 705-717, Korea.
Tel.: 82-53-620-4365
Fax: 82-53-653-6628
E-mail: dwjeong@ynu.ac.kr
J. Cell. Mol. Med. Vol 15, No 10, 2011
2083
© 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
phenomena are known to be achieved by excessive bone resorp-
tion and reduced bone formation during bone remodelling,
respectively [2].
Recent progress in stem cell biology has provided a promis-
ing strategy for treatment of multiple degenerative disorders [3].
In particular, adult stem cells have emerged as an important issue
due to the potential for use of their
ex vivo
expanded progenies in
cell-based regenerative medicine, tissue engineering and cancer
therapy [4]. Adult stem cells participate in replenishment of cells
that are lost during regeneration of damaged tissue, as well as in
normal tissue development. MSCs, which possess unique
immunosuppressive and anti-inflammatory properties and a
capacity for homing to injured tissues, have been isolated from
various tissues, including bone marrow, adipose, hair follicles,
spleen, placenta, umbilical cord blood, foetal liver and lung [4].
MSCs obtained from adipose tissues could constitute a promis-
ing source of cells for use in cell-based therapy and tissue
engineering [5]. Therapeutic potential for bone regeneration by
systemic transplantation of genetically manipulated MSCs
co-expressing C-X-C chemokine receptor type 4 (CXCR4) and
Cbfa-1 in glucocorticoid-induced osteoporotic mice has been
recently suggested [6].
The main benefits of adipose-derived stem cells (ASCs) in
therapeutic applications, as compared with bone marrow-
derived MSCs, are that adipose tissue is readily accessible and
relatively abundant, and the stem cell population can be easily
harvested by simple methods, such as lipoaspiration or surgical
resection, and can be rapidly expanded
ex vivo
[7]. ASCs have
also been shown to support differentiation of haematopoietic
progenitors into myeloid and B lymphoid cells [8]. ASC-derived
cellular therapy has been investigated with respect to a wide
variety of human diseases, such as skeletal muscle disorders,
cardiovascular disorders and diabetes mellitus, and in bioengi-
neering for tissue regeneration [4]. Additive support of ASCs in
tissue repair and regeneration has been reported to include dif-
ferentiation into a proper cell lineage and paracrine mechanisms
mediated by secreted cytokines and growth factors [5]. Growing
evidence indicates that paracrine factors play a critical role in
ASC-induced tissue repair [9, 10]. Bioactive levels of multiple
paracrine factors, including hepatocyte growth factor (HGF),
vascular endothelial growth factor, nerve growth factor, insulin-
like growth factor-1 (IGF-1), transforming growth factor-,
basic fibroblast growth factor and granulocyte macrophage
colony-stimulating factors are known to be released by ASCs
[11]. Moreover, conditioned media obtained from ASCs have
been found to protect against cerebellar granule neuron
apoptosis [12].
Considering the fact that the pathogenetic mechanisms
underlying osteoporosis cover multiple sets of dynamic param-
eters, a systemic approach using stem cell transplantation is
attractive for treatment of osteoporosis. In the present study,
we investigated the question of whether systemically trans-
planted human ASCs (hASCs) could restore bone function and
structure in an ovariectomized (OVX)-induced osteoporotic
mouse model.
Materials and methods
Isolation and culture of hASCs
Human subcutaneous adipose tissues were obtained under approval
from the institutional review board of Yeungnam University Medical
Center. The lipoaspirate was incubated with collagenase type I solution
(Worthington Biochemical, Lakewood, NJ, USA) for 1 hr at 37C, and fil-
tered through 500 and 250 m filters. Following centrifugation, the stro-
mal vascular fraction was resuspended in DMEM (HyClone, Logan, UT,
USA) supplemented with 10% foetal bovine serum (FBS) (HyClone), 100
U/ml penicillin and 100 g/ml streptomycin. Cells were cultured under a
humidified atmosphere of 5% CO
2
at 37C and used for experiments at
passages 3 to 5.
Preparation of conditioned media
hASCs and human embryonic kidney (HEK)293T fibroblast cells were
seeded on a 100 mm dish in DMEM supplemented with 10% foetal bovine
serum. At 90% confluence, cells were washed three times with phosphate-
buffered saline and the medium was replaced with serum-free medium.
After 1 hr, the medium was removed and fresh serum-free medium was
added. As a control, conditioned media from hASCs (hASC-CM) and
HEK293T cells (HEK293T-CM) were collected at 60 and 24 hrs after
culture, respectively, which did not cause cell death for the indicated times
(data not shown). Media were centrifuged at 300
g
for 5 min., and
filtered through a 0.22 m syringe filter. For
in vitro
cultures of osteoblasts
and osteoclasts in hASC-CM, equal volumes of hASC-CM and -MEM
(HyClone) with FBS adjusted to 10% were used without concentration
(50% hASC-CM). HEK293T-CM/-MEM (1:1) or DMEM/-MEM (1:1)
was used as a control medium. For Western blot analysis and mass
spectrometric identification of secreted proteins in hASCs, hASC-CM were
concentrated 50-fold using an Amicon Ultra-15 (Millipore, Bedford, MA,
USA) with a 10,000 molecular weight cutoff.
RT-PCR, Western blotting
and ELISA assay
For detection of mRNA level, total RNA was isolated with TRIzol
(Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruc-
tions. About 2 g of total RNA was reverse transcribed using Moloney
murine leukaemia virus reverse transcriptase (Promega, Madison, WI,
USA) with oligo dT at 42C for 1 hr. Detailed information on PCR,
including primer sequences and cycles, is provided in Table S1. For
Western blotting, cells were lysed by addition of lysis buffer containing
20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate,
1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% SDS, protease
inhibitors (Complete tablets, Roche Molecular Biochemicals, Mannheim,
Germany), 1 mM Na
3
VO
4
and 1 mM NaF. Proteins were separated by 10%
SDS-PAGE, transferred to a nitrocellulose membrane and probed with
specific antibodies. All antibodies used in this study are described in the
Supporting Information. Protein levels of HGF in serum were measured
using the Quantikine ELISA Kit (R&D Systems, Inc., Minneapolis, MN,
USA). Urine deoxypyridinoline (DPD) was assayed by competitive
enzyme immunoassay using the MicroVue DPD EIA kit (Quidel
Corporation, Santa Clara, CA, USA).
2084 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
Protein identification using mass spectrometry
As previously described, proteins obtained from SDS-PAGE gels or from
concentrated hASC-CM were identified using mass spectrometry (Gwangju
Center, Korea Basic Science Institute, Korea) [13]. Detailed methods are
described in the Supporting Information.
Microcomputed tomography and histological
analysis of bone
A total of 2 10
6
cells were injected into sham-operated or OVX female
ddY mice (8 weeks old, Central lab animal, Korea)
via
tail vein on post-
operative day 4 and killed at day 24 after injection (
n
6 per group).
Microcomputed tomography (CT) and histological analysis were
performed, as reported previously [14, 15]. Trabecular morphometry within
the proximal tibia was quantified using high resolution CT (Skyscan
1076 CT, Aartselaar, Belgium). From CT data, bone loss indices, includ-
ing bone volume/total volume (BV/TV), trabecular number (Tb.N) and bone
mineral density (BMD) were assessed. For analysis of bone formation, mice
were injected with calcein (10 mg/kg) on post-operative day 14 and day 21
and were killed at post-operative day 24. For histological evaluation, serial
5-m-thick sagittal sections were made using a microtome. Haematoxylin
and eosin staining was used for detection of osteoblasts and tartrate resist-
ant acid phosphatase (TRAP) staining was used for visualization of osteo-
clasts. Histological images were analysed by an Aperio ScanScope Model
T3 and ImageScope software (Aperio Technologies, Inc., Vista, CA, USA).
All animal procedures were approved by the institutional review board of
Yeungnam University Medical Center and were in accordance with the Guide
for the Care and Use of Laboratory Animals.
Detection of hASCs after intravenous injection
After 8-week-old female ddY mice had been OVX, hASCs (2 10
6
cells/
mouse) labelled with iron oxide (Ferridex, Berlex Laboratories, Inc., Wayne,
NJ, USA) were injected into OVX mice
via
tail vein on post-operative day 4
and killed at day 24 after injection. Bone tissues were fixed and decalcified
with EDTA solution. After washing, paraffin-embedded specimens were
sectioned, deparaffinized, incubated with 1% potassium ferrocyanide in
1% HCl for 30 min. and counterstained with nuclear fast red. Six animals
were included in each group.
In vitro
assays of osteoblast differentiation,
adhesion and spreading
Primary osteoblast cells were prepared from the calvaria of 3-day-old
C57BL/6J mice (Central lab animal) and cultured with -MEM containing
antibiotics and 10% FBS at 37C in 5% CO
2
. Osteoblastic differentiation in
the absence or presence of hASC-CM and/or 1,25-dihydroxyvitamin D
3
(VitD
3
) was monitored for 24 days. For mineralization assay, we used 1
10
4
cells per well in 48-well plates. For quantification of calcium, mineralized
osteoblasts were decalcified with 0.6 M HCl and calcium ion concentration
was determined using a QuantiChrom™ calcium assay kit (DICA-500,
BioAssay Systems, Hayward, CA, USA), according to the manufacturer’s
procedures. In order to normalize this assay, the remaining cells were
lysed in 0.1 M NaOH/0.1% SDS, and protein concentration was determined
using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA).
Detailed methods for cell adhesion and spreading assays are described
in Supporting Information.
In vitro
assays of osteoclast differentiation
and function
Bone marrow-derived monocytes were isolated from the tibia and femur of
6-week-old C57BL/6J mice (Central lab animal) by flushing the bone
marrow cavity. Osteoclast precursors were prepared as described [16] and
detailed methods are described in the Supporting Information. To induce
osteoclast differentiation, osteoclast precursors were cultured with 5 ng/ml
of macrophage colony-stimulating factor (M-CSF) for 12 hrs, and the
medium was then replaced with either control medium (50% HEK293T-CM
in -MEM or 50% DMEM in -MEM) or 50% hASC-CM (50% hASC-CM in
-MEM) in the absence or presence of M-CSF (30 ng/ml) or receptor
activator for nuclear factor B ligand (RANKL, 50 ng/ml) for the indicated
time periods. When cultured in 50% hASC-CM in -MEM, the final
concentration of FBS was adjusted to 10%. For positive controls, osteo-
clast precursor cells were cultured with M-CSF (30 ng/ml) and RANKL
(100 ng/ml) for 3 to 4 days. For identification of osteoclasts, TRAP stain-
ing was performed with an Acid Phosphatase Kit (Sigma-Aldrich, St. Louis,
MO, USA), according to the manufacturer’s instructions. A TRAP solution
assay was performed by addition of 5.5 mM
P
-nitrophenyl phosphate,
a colorimetric substrate, in the presence of 10 mM sodium tartrate at
pH 5.2. The reaction product was quantified by measurement of optical
absorbance at 405 nm.
Statistical analysis
Data are presented as means S.D. from at least three independent exper-
iments. Differences were considered statistically significant if
P
-value was
less than 0.05. Statistical analyses were performed with the two-tailed
Student’s t-test for analysis of differences among groups.
Results
Characterization of hASCs
Cell surface marker expression by hASCs isolated from human
adipose tissue was characterized. Flow cytometric analysis of
hASCs revealed that high levels of MSC-related antigens CD13,
CD29, CD44, CD90 and CD166 were expressed; however,
haematopoiesis-related antigens CD31, CD34, CD117 and human
leucocyte antigen CD45 were not expressed (Fig. S1). In addi-
tional, the ability of hASCs to differentiate into osteoblast, chon-
drocyte and adipocyte lineages at passage 3 was experimentally
confirmed by von Kossa staining, Alcian blue staining and Oil red
O staining, respectively (Fig. S2). These results showed that
hASCs have multilineage potential and characteristics of MSCs.
J. Cell. Mol. Med. Vol 15, No 10, 2011
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Gene and protein expression of bone-related
factors in hASCs and mass spectrometric
identification of proteins secreted from hASCs
It is well established that hASCs have the ability to secrete a
variety of cytokines, including angiogenic, anti-apoptotic and pro-
inflammatory factors [11]. In this study, we examined expression
and secretion of bone-related growth factors and cytokines in
hASCs. Using RT-PCR, we observed expression of genes encod-
ing HGF, M-CSF, bone morphogenetic protein 2 (BMP-2), BMP-4,
osteopontin, RANKL and tumour necrosis factor- in hASCs
(Fig. 1A). Secretion of RANKL and M-CSF, which are essential for
osteoclastogenesis, and HGF, which is known to increase DNA
synthesis and proliferation of osteoblasts and osteoclasts [17],
were confirmed in hASC-CM using Western blotting and ELISA
(Fig. 1B and C).
To determine whether other proteins that affect bone function
were present in hASC-CM, the secretome of primary hASC cultures
was analysed using both gel-based and non-gel based approaches.
First, SDS-PAGE combined with matrix-assisted laser desorption/
ionization time-of-flight/time-of-flight mass spectrometry was
used for protein identification in hASC-CM. Tryptic peptides from
each band were identified using peptide mass fingerprinting and
peptide sequencing, identifying 33 proteins in hASC-CM (Table
S2). Second, we subjected the proteins of hASC-CM to in-solution
tryptic digestion and nanoLC-tandem mass spectrometry, and
identified 23 proteins in hASC-CM (Table S3). Table 1 shows a list
of a total of 43 proteins identified by gel- and non-gel-based meth-
ods. Thirty-three proteins (77%) contained a predicted secretion
signal peptide, and several proteins were reported to be involved in
bone function. For example, secreted protein acidic and rich in
cysteine (SPARC), periostin and fibronectin are known to stimulate
proliferation and differentiation of osteoblasts [18–20];
2
-microglobulin and thrombospondin-1 are involved in stimula-
tion of osteoclast formation and function [21, 22]. The presence of
some proteins identified in mass spectrometric analysis, such as
periostin, plasminogen activator inhibitor 1 and SPARC was
validated using Western blotting (Fig. 1B). Combined results from
RT-PCR, Western blotting, ELISA and mass spectrometric protein
identification demonstrated the release by hASCs of a variety of
proteins and cytokines related to bone function, suggesting that
paracrine factors produced by hASCs might possibly play an
important role in bone remodelling and repair.
Effects of systemic transplantation of hASCs
on OVX-induced bone loss
We performed CT analysis to determine the impact of hASCs on
OVX-induced osteoporotic mice (Fig. 2A). Systemic transplanta-
tion of hASCs into OVX mice prevented OVX-induced bone loss in
mice. When compared with OVX mice, bone loss indices, includ-
ing bone volume fraction, trabecular number and BMD in hASC-
transplanted OVX mice were restored to normal. The recovery
effect of hASCs on bone loss was confirmed by measurement of
the concentration of urinary DPD, a useful marker of bone resorp-
tion. Compared with OVX mice, a significantly lower level of uri-
nary DPD was exhibited by hASC-transplanted OVX mice (Fig. 2B).
Mineral apposition rate and bone formation rate, as measured by
calcein label-based analysis, were restored to the level of sham-
operated mice in hASC-transplanted OVX mice (Fig. 2C). Of partic-
ular interest, histological analysis showed that the numbers of
both osteoblasts and TRAP
osteoclasts adhering to trabecular
bone surfaces were increased in hASC-transplanted OVX mice
compared with sham and control OVX mice (Fig. 2D), suggesting
that systemic transplantation of hASCs in OVX mice might affect
proliferation or differentiation of osteoblasts and osteoclasts.
HGF that play important roles in bone formation and remodel-
ling [17, 23] were expressed by hASCs (Fig. 1). Consistently,
serum level of human HGF was markedly higher in hASC-trans-
planted OVX mice than in control OVX mice (Fig. 2E). Because
oestrogen plays a critical role in OVX-induced bone loss [24], we
explored the ovarian and uterine status of recipient mice (Fig. S3).
Oestrogen-deficient OVX mice showed decreases in uterine weight
and diameter. No significant differences in ovarian and uterine
Fig. 1 Gene expression analysis of bone-related factors in hASCs and pro-
tein identification in hASC-CM. (A) mRNA levels for bone-related genes in
hASCs. The mRNA expression profile of genes representative of osteoblast
and osteoclast function was analysed using RT-PCR. The level of glycer-
aldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal con-
trol for equal loading, and a PCR reaction without specific primers was
used as a negative control. The gel is representative of three independent
experiments. HGF: hepatocyte growth factor; M-CSF: macrophage colony-
stimulating factor; BMP-2: bone morphogenetic protein 2; BMP-4: bone
morphogenetic protein 4; OPN: osteopontin; RANKL: receptor activator for
nuclear factor B ligand; TNF-: tumour necrosis factor-. (B) Protein
expression levels of bone-related genes. Levels of RANKL, M-CSF, periostin,
plasminogen activator inhibitor 1 (PAI-1) and SPARC in hASC-CM concen-
trated 50-fold were determined by Western blot analysis. C: control
medium; CM: hASC-CM. (C) Secretion of HGF from hASCs was measured
by ELISA. Data are normalized as pg per 10
6
cells and are expressed as
means S.D. (
n
3). ND: not detected.
2086 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
status were observed in hASC-transplanted OVX mice and control
OVX mice. From these data, we confirmed the effectiveness of our
OVX surgeries and the oestrogen status in the OVX mice.
For detection of hASCs in OVX mice after systemic transplan-
tation, hASCs labelled with iron oxide were transplanted into OVX-
induced osteoporotic mice
via
tail vein injection on post-operative
day 4 and mice were killed on day 24 after transplantation.
Distribution of hASCs was assessed by Prussian blue staining.
Iron-labelled transplanted cells were detected in the periosteum of
recipient OVX mice (Fig. 2F).
Table 1 Mass spectrometric identification of proteins secreted by hASCs
*SignalP indicates the probability score calculated with SignalP software.
Protein (accession number) SignalP* Bone-related function Protein (accession number) SignalP Bone-related function
Fibronectin (Q9UMK2) 0.997
Osteoblast survival and
differentiation [20]
Collagen
1
(I) chain (Q9UML6)
0.999
Osteoblast maturation
and differentiation [42]
Collagen
2
(I) chain (P08123)
0.997
Osteoblast maturation and
differentiation [42]
Thrombospondin-2 (P35442) 1.000
Osteoblast differentiation
[43]
Collagen
1
(III) chain (P02461)
0.999
Collagen
1
(VI) chain (P12109)
1.000
SR calcium ATPase 1 (O14983) Thrombospondin-1 (P07996) 0.994 Osteoclast function [22]
-actinin-1 (P12814)
Protein FAM40A (Q5VSL9)
Periostin (Q15063) 0.999
Osteoblast proliferation
and differentiation [19]
ig-h3 (Q15582)
1.000
Osteoblast adhesion and
differentiation [44]
Thrombospondin-5 (P49747) 1.000
Chondrocyte proliferation
[45]
Matrix metalloproteinase 1
(P03956)
1.000
Osteoblast differentiation
[46]
72 kD type IV collagenase (P08253) 1.000
Bone cell growth and
proliferation [47]
Plasminogen activator
inhibitor 1 (P05121)
0.999
Bone mineralization and
bone growth [48]
Albumin (P02768) 1.000 Moesin (P26038)
Galectin-3-binding protein
(Q08380)
1.000
Protein disulfide-isomerase
(P07237)
1.000
Fibulin-3 (Q12805) 0.999 Vimentin (P08670)
EF-1-alpha 1 (P68104) Serpin A12 (Q8IW75) 0.997
Cathepsin L1 (P07711) 0.999 Bone resorption [49] Cathepsin D (P07339) 1.000 Osteoblast calcification [50]
Glia-derived nexin (P07093) 0.995
Pentraxin-related protein
PTX3 (P26022)
1.000
-actin (P60709)
Decorin (P07585) 1.000
Follistatin-related protein 1 (Q12841) 1.000 Sulfhydryl oxidase 1 (O00391) 1.000
IGF-binding protein 7 (Q16270) 0.998 Cathepsin B (P07858) 1.000
Peptidyl-prolyl cis-trans
isomerase B (P23284)
0.863 SPARC (P09486) 1.000
Osteoblast formation,
maturation and survival
[18]
Lumican (P51884) 1.000 Bone formation [51]
Metalloproteinase inhibitor 2
(P16035)
1.000
Osteoblast differentiation
[52]
Transgelin (Q01995) Peroxiredoxin-1 (Q06830)
Metalloproteinase inhibitor 1
(P01033)
Bone turnover [53] Protein S100-A6 (P06703)
2
-microglobulin (P61769)
1.000 Osteoclast formation [21]
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2088 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
The effect of hASC-CM on osteoblast
and osteoclast differentiation
Our results suggested that hASCs expressed and secreted bone-
activating factors into the extracellular space, which are able to
potentiate osteoblast and osteoclast differentiation (Fig. 1 and
Table 1). In differentiation of osteoblasts and osteoclasts
in vitro
,
it is established that VitD
3
and prostaglandin E2 can lead to
osteoblast activation and mineralization and that RANKL, in com-
bination with M-CSF, can induce multinucleated osteoclast forma-
tion from a macrophage lineage [14, 25, 26]. Thus, we assessed
a stimulatory action of hASC-derived secreted factors in
osteoblast and osteoclast differentiation under various combined
cultures of bone cell-stimulating factors, hASCs-CM and
HEK293T-CM as a control. Among various combinations,
osteoblast cultures with hASC-CM and VitD
3
displayed mineral
deposition, but did not with HEK293T-CM and VitD
3
(Fig. 3A). In
additional, combined treatment of hASC-CM- and RANKL-induced
differentiation of osteoclast precursors into TRAP
multinucleated
cells (MNCs), whereas HEK293T-CM and RANKL treatment failed
to induce their differentiation (Fig. 3B). These results indicate that
osteoblastogenic factors present in hASC-CM can be substituted
for prostaglandin E2, and that osteoclastogenic factors in hASC-
CM can be substituted for M-CSF.
Stimulatory effect of hASC-CM on osteoblast
proliferation and differentiation
Compared with sham-operated and OVX mice, systemic trans-
plantation of hASCs into OVX mice resulted in an increase in the
number of osteoblasts and osteoclasts on bone surfaces (Fig. 2D).
To elucidate the molecular mechanisms responsible for
in vivo
results, we investigated the question of whether hASC-CM could
affect the physiological properties of osteoblasts, including prolif-
eration, adhesion, spreading and differentiation. We found that
hASC-CM induced an increase in the osteoblast population, com-
pared with the control medium (Fig. 4A). We also found that
hASC-CM markedly promoted cell attachment and spreading,
compared with the control medium (Fig. 4B and C). These
enhanced activities of cell attachment and spreading were com-
pletely inhibited by EDTA, but not by heparin, suggesting that cell
attachment and spreading in osteoblasts might be promoted by
hASC-CM through a divalent cation-dependent mechanism.
Whereas cultures supplemented with either hASC-CM or VitD
3
alone did not induce matrix mineralization of osteoblasts, addition
of both hASC-CM and VitD
3
to cultures accelerated the mineraliza-
tion of osteoblasts (Fig. 4D).
We next examined the effects of hASC-CM on signalling events
in osteoblasts. As shown in Figure 4E, short-term treatment of
osteoblasts with hASC-CM resulted in increased phosphorylation
of receptor-regulated Smad 1/5/8, which is known to function as
an intracellular mediator of osteoblast differentiation signalling
[27], and to play an important role in early osteoblastogenesis [2].
However, neither phosphorylation nor expression levels of -
catenin in osteoblasts were altered by treatment with hASC-CM. In
addition, we measured the activities of three MAPKs, ERK, c-jun
NH
2
-terminal kinase (JNK) and p38 in osteoblasts after long-term
treatment with hASC-CM. Maximal activation of both ERK and
JNK, which can, in part, stimulate osteoblast differentiation [28]
was induced by day 3 after treatment with hASC-CM however, p38
was not affected (Fig. 4F).
Stimulatory effect of hASC-CM on osteoclast
precursor survival and osteoclast differentiation
The combination of M-CSF and RANKL is indispensable for both
survival and differentiation of osteoclast precursors [14]. To
evaluate the effect of hASC-CM on survival and proliferation of
osteoclast precursors, we counted the numbers of osteoclast pre-
cursors after exposure to hASC-CM. The number of osteoclast
precursors cultured with hASC-CM was considerably higher than
that of osteoclast precursors cultured with control medium
(Fig. 5A). To determine whether hASC-CM could stimulate osteo-
clast differentiation, osteoclast precursors were cultured under the
control medium or hASC-CM in the presence or absence of M-CSF
or RANKL and stained with TRAP to detect mature osteoclasts.
TRAP
MNCs were not detected in osteoclast precursors cultured
Fig. 2
In vivo
effects of hASCs on OVX-induced osteoporotic mice. (A) CT analysis of bone tissue. 3D reconstruction of tibiae from sham-operated
(Sham) and OVX mice transplanted without or with hASCs (OVX and OVX hASC) was analysed by CT. Scale bar: 0.5 mm. Histograms represent 3D
trabecular structural parameters in tibia: bone volume/total volume (BV/TV), trabecular number (Tb.N) and BMDs. Data represent mean S.D. (
n
6).
(B) Urinary samples were obtained prior to killing and levels of DPD were measured by ELISA. Data represent mean S.D. (
n
3). (C) Mineral apposition
rate and bone formation rate (BFR/BS) were measured by calcein labelling. Data represent mean S.D. (
n
3). Scale bar: 10 m. (D) Histological analysis
of tibiae from OVX mice systemically transplanted with or without hASCs. Osteoblasts and osteoclasts on the trabecular bone surface were visualized by
haematoxylin and eosin and TRAP staining, respectively. The number of osteoblasts (NOb/BS) and osteoclasts (NOc/BS) is expressed as a cell number
per mm of trabecular bone surface. Scale bar: 100 m. Data represent mean S.D. (
n
6). The level of human HGF in serum of sham-operated or OVX
mice with or without systemic transplantation of hASCs was determined by ELISA. (E) Data represent mean S.D. (
n
3). (F) Detection of hASCs in
tibia of recipient OVX mice after systemic transplantation. hASCs (2 10
6
cells/200 l) labelled with iron oxide were injected into OVX-induced osteoporotic
mice
via
tail vein on post-operative day 4 and killed at day 24 after injection. Tissue sections from tibia of recipient mice were subjected to Prussian blue stain-
ing. Results shown are representative of three experiments. Scale bar: 50 m. a:
P
0.05; b:
P
0.01; c:
P
0.001. 140 169 mm (300 300 DPI).
J. Cell. Mol. Med. Vol 15, No 10, 2011
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Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
in hASC-CM in the presence or absence of M-CSF. However,
osteoclast precursors cultured in hASC-CM in the presence of
RANKL formed much higher numbers of TRAP
MNCs at day 9 of
culture (Fig. 5B), consistent with the result shown in Figure 3B,
suggesting that hASC-CM could efficiently support osteoclast for-
mation in a manner similar to that of M-CSF. TRAP activity was
also higher in osteoclast precursors cultured with hASC-CM than
in osteoclast precursors cultured with control medium (Fig. 5C).
mRNA expression of osteoclastic markers, such as cathepsin K,
TRAP, NFATc1 and matrix metalloproteinase-9 were induced when
osteoclast precursors were cultured in hASC-CM (Fig. 5D). All
three major subfamilies of MAPKs, ERK, JNK and p38, which are
known to play an important role in osteoclastogenesis, were
activated immediately after hASC-CM treatment in osteoclast
precursors (Fig. 5E). In contrast to the stimulatory action of
hASC-CM in osteoblast adhesion and spreading (Fig. 4B and C),
hASC-CM did not affect adhesion and spreading of osteoclast
precursors (data not shown).
Discussion
Bioactive trophic factors secreted by ASCs have been reported to
directly contribute to angiogenic and anti-apoptotic effects in
ischemic limb disease, restoration of heart function and nerve
sprouting following myocardial infarction, and skin wound healing
[9, 10, 29, 30]. In this study, we showed that hASC-based therapy
via
systemic transplantation could be effective in bone repair by a
mechanism predominantly mediated through secretion of
paracrine factors by hASCs.
Osteoporosis is a chronic and complex disease involving an
uncoupling between bone formation and bone resorption. Multiple
pathogenetic mechanisms are involved in loss of bone mass and
microarchitectural reduction of bone tissue in osteoporosis [1].
Bone repair is known to be a complex physiologic process that is
regulated by several cell types, as well as the extracellular matrix
and growth factors [31]. Secretion of important growth factors
with potential for mediation of bone repair, including vascular
endothelial growth factor, IGF-1 and TGF- [32] from ASCs has
been demonstrated [11]. In addition, we found that hASCs could
express and secrete various cytokines, growth factors and pro-
teins that are required for bone function and remodelling. These
included M-CSF, RANKL, BMP-2, BMP-4, HGF and bone-related
extracellular matrix proteins. Our
in vivo
results revealed that
OVX-induced bone loss was restored by systemic transplantation
of hASCs into recipient OVX mice. We further showed that
hASC-injected OVX mice exhibited an increase in the number of
both osteoblasts and osteoclasts. This could be explained by the
balance between bone resorption and bone formation. The levels
of bone resorption by osteoclasts did not exceed those of bone
formation by osteoblasts, resulting in a net increase of bone
mass. These findings indicate that hASCs can rescue oestrogen
Fig. 3 Effects of hASC-CM on osteoblast and osteoclast differentiation. (A) Osteoblast mineralization. The mineralized extent of osteoblasts was quanti-
fied at day 24 after incubation of cells in 50% hASC-CM or 50% HEK293T-CM (as a control media) in the presence or absence of 10 nM VitD
3
. For a
positive control, osteoblasts were cultured in the presence of 100 g/ml ascorbic acid and 10 mM -glycerophosphate. Data represent mean S.D.
(
n
3). (B) Osteoclast differentiation. Osteoclast precursors (5 10
4
cells per well in 48-well plates) were cultured for 8 days in 50% hASC-CM or 50%
HEK293T-CM in the presence or absence of RANKL (50 ng/ml), as indicated. Cells were stained with TRAP and TRAP
MNCs (3 nuclei) were counted
under a light microscope. Representative images of three independent experiments are presented. Scale bar: 100 m. b:
P
0.01; c:
P
0.001.
2090 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
deficiency induced bone loss by simultaneous stimulation of
osteoblast-mediated bone formation and osteoclast-mediated
bone resorption in recipient OVX mice.
Stem cell homing capacity to the site of tissue injury is impor-
tant for effective stem cell therapy. When hASCs labelled with
Qtracker-delivered quantum dots were injected into OVX mice on
post-operative day 28, a quantum dot signal could not be detected
in bone at 60 min. after injection (Fig. S4A). A control experiment
was performed with an experimental ischemia model in rat kidney
to evaluate our system for assessment of the distribution of
hASCs. Intense fluorescence from Qtracker labels and iron-
labelled cells could be clearly seen at the ischemic site after injec-
tion of quantum-dot labelled or iron-labelled hASCs into ischemic
kidney rats
via
tail vein (Fig. S4B and C). These results demon-
strate that hASCs can move promptly into the damaged site in an
acute ischemia-induced kidney model, but were not able to move
into bone of OVX-induced osteoporotic mice. We made a profound
attempt to identify localization of transplanted hASCs in a whole
tissue. Among various tissues, tissue sections made from tibias of
OVX mice following intravenous administration of hASCs showed
that the transplanted cells were localized in the periosteum, but
not in cortical bone. Periosteum is located at the outer bone sur-
face along the periosteal cortex of cortical bone, and periosteal
bone apposition plays a critical role in skeletal development [33].
Periosteum has been reported to show a dramatic response to
bone growth factors with a significant increase in new bone for-
mation. However, the question of whether or not the position of
hASCs on periosteum specifically targeted a nearby part of the
damaged bone is obscure. Our findings suggest that hASCs might
not be directly involved in transdifferentiation into osteoblasts in
trabecular and cortical bones. Instead, secreted factors from
hASCs that are located sporadically all over the body and/or
located in periosteum of the damaged bone tissue might be impli-
cated in bone regeneration.
Synthesis of VitD
3
has been reported to occur in bone cells
[34], and stimulation of osteoblast matrix mineralization by HGF in
concert with VitD
3
has also been reported [35]. Our study showed
that hASC-CM together with VitD
3
-induced mineralization of
Fig. 4 Stimulatory action of hASC-CM in osteoblast proliferation, adhesion, spreading and differentiation. (A) Osteoblast proliferation. Following culture
of osteoblasts (2 10
4
cells per well in 24-well plates) using a control medium (50% DMEM in -MEM) or hASC-CM (50% hASC-CM in -MEM) for
the indicated times, trypan blue-excluded cells were counted using a haemocytometer under a light microscope. Data represent mean S.D. (
n
3).
Cell adhesion (B) and spreading (C) assays were performed on osteoblasts incubated for 1 hr in the same media as in (A). For assessment of cell adhe-
sion, cells were stained with crystal violet, dissolved in 2% SDS, and absorbance was measured at 595 nm. For assessment of cell spreading, cell area
was measured using Image-Pro plus software. In inhibition experiments, cells were pre-incubated with either 5 mM EDTA or 100 g/ml heparin at
37C for 10 min. Values are expressed as the mean S.D. (
n
3). (D) Calcium content in osteoblasts. The extent of mineralization of osteoblasts (1
10
4
cells per well in 48-well plates) was quantified at day 24 after incubation of cells in the presence or absence of hASC-CM and/or 10 nM VitD
3
. For a
positive control, osteoblasts were cultured in the presence of 100 g/ml ascorbic acid and 10 mM -glycerophosphate. Data represent mean S.D.
(
n
3). (E) and (F) Osteoblast-stimulating signals. Osteoblasts were stimulated with 50% hASC-CM for the indicated times and activation of Smad 1/5/8,
-catenin, ERK, JNK and p38, which are implicated in osteoblast differentiation and function, was assessed by Western blotting with specific antibodies.
Numbers indicate the ratios of phosphorylated MAPKs to total MAPKs. -actin was used as a loading control. Data are representative of three independent
experiments. b:
P
0.01; c:
P
0.001.
J. Cell. Mol. Med. Vol 15, No 10, 2011
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Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
osteoblast cells, indicating the possibility that the combination of
HGF secreted from hASCs and VitD
3
produced by bone cells plays
an important role in osteoblast differentiation
in vivo
. Cellular
adhesion to extracellular matrix is known to be essential to
osteoblast survival, proliferation and differentiation [20]. In our
experiments, cell attachment and spreading in osteoblasts were
markedly increased by treatment with hASC-CM, and those
increased activities were attenuated in the presence of EDTA,
demonstrating that hASC-CM promotes adhesion and spreading
of osteoblasts in a divalent cation-dependent manner. Integrins
expressed on the plasma membrane of osteoblasts are known to
require extracellular divalent cations in order to bind to their lig-
ands [36], and interaction of integrin with extracellular matrix
components activates osteoblast survival and matrix mineraliza-
tion [20]. Therefore, various extracellular matrix proteins in hASC-
CM, including fibronectin and type I collagen are likely to partici-
pate not only in cell adhesion, but also in proliferation and differ-
entiation of osteoblast cells
via
integrin signalling.
Because M-CSF and RANKL are known to be critical for
survival and differentiation of osteoclasts, and were detected in
hASC-CM, we expected that survival and differentiation of osteo-
clast precursors might be induced by hASC-CM. Indeed, we found
that hASC-CM was able to maintain the survival of osteoclast pre-
cursors. However, hASC-CM failed to direct the differentiation of
osteoclast precursors into TRAP
MNCs. Compared with precursors
cultured in control medium and RANKL, osteoclast precursors
cultured in hASC-CM and RANKL formed much higher numbers of
TRAP
MNCs. In the presence of RANKL, HGF has been shown to
support osteoclast differentiation in a manner similar to that of
M-CSF [37]. In addition, HGF receptor was expressed by both
Fig. 5 Stimulatory action of hASC-CM in survival and differentiation of osteoclast precursor cells. (A) Osteoclast precursor survival. Osteoclast precur-
sors (2 10
5
cells per well in 6-well plates) were cultured in the presence of 50% hASC-CM for the indicated times and trypan blue-excluded viable cells
were then counted using a haemocytometer. Data represent mean S.D. (
n
3). (B) Osteoclast differentiation. Osteoclast precursors (5 10
4
cells per
well in 48-well plates) were cultured for 8 days in control medium or in 50% hASC-CM in the presence or absence of M-CSF (30 ng/ml) or RANKL
(50 ng/ml), as indicated. Media were replenished on day 2. After 8 days, cells were stained with TRAP and the number of TRAP
MNCs (3 nuclei) was
counted. Data represent mean S.D. (
n
3). Representative images of three independent experiments are presented. Scale bar: 100 m. (C) TRAP
activity. Osteoclast precursors (2 10
5
cells per well in 6-well plates) were incubated for the indicated times in control medium or in 50% hASC-CM and
subjected to the TRAP assay. Data are representative of three independent experiments and expressed as mean S.D. (
n
3). (D) Osteoclast-specific
gene expression. Osteoclast precursors were cultured in 50% hASC-CM for 2 days and the mRNA level for osteoclast marker genes was then determined
using RT-PCR. Data are representative of three independent experiments. (E) Osteoclast-stimulating signals. Following adaption of osteoclast precursors
for 12 hrs in the presence of M-CSF (30 ng/ml) and further incubation without M-CSF for 6 hrs, cells were stimulated with control medium or 50% hASC-CM
for the indicated times and immediate responding signals in osteoclast precursors were analysed by Western blotting with specific antibodies to p-ERK,
p-JNK and p-p38. -actin was used as a loading control. The gel is representative of three independent experiments. b:
P
0.01; c:
P
0.001.
2092 © 2011 The Authors
Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
osteoblasts and osteoclasts [17]. Therefore, our findings sug-
gest that HGF, or M-CSF, or both in hASC-CM might be able to
support proliferation and differentiation of osteoclast precur-
sors, but that the level of RANKL in hASC-CM was not sufficient
to induce osteoclastogenesis
in vitro
. These results are consis-
tent with those of a previous report showing that ASCs could
support differentiation of haematopoietic progenitor cells [8].
Involvement of ERK in osteoclast survival has been reported
[38], and involvement of p38 and JNI in osteoclast differentia-
tion has also been reported [39, 40]. ERK, JNK and p38 were
strongly activated in response to hASC-CM in osteoclast precur-
sors, implying that paracrine factors released by hASC could
transiently activate all three major MAPKs (ERK, JNK and p38)
that contribute to induction of osteoclastogenesis. In addition,
hASC-CM induced maximal activation of ERK and JNK at day 3
after osteoblast culture. Taken together, these results suggest
that enhanced survival and differentiation of osteoblasts and
osteoclasts in cultures containing hASC-CM were mediated, at
least in part, by activation of MAPKs.
Key mechanisms by which ASCs might repair and regenerate
damaged tissues have been suggested to include paracrine
effects of secreted cytokines and growth factors, and direct dif-
ferentiation to a desired cell lineage [5]. In a developing mouse
model, differentiation into osteoblasts appears to be especially
important for treatment of osteogenesis imperfecta [41].
However, hASC-induced improvement of bone mass could not
be fully explained by direct differentiation into osteoblasts
because hASC-injected OVX mice exhibit increased numbers of
both osteoblasts and mature osteoclasts, and hASC-CM simul-
taneously stimulates proliferation and differentiation of both
osteoblasts and osteoclasts
in vitro
. Based on
in vivo
and
in vitro
results from our adult mouse model, we suggest that
paracrine effects induced by systemic transplantation of hASCs
are the predominant mechanism mediating the therapeutic
effects of hASCs in treatment of osteoporosis. Collectively,
hASCs transplanted
via
the circulatory system could function as
antiresorptive and anabolic agents, and could be a valuable
therapeutic option for treatment of both high- and low-turnover
osteoporosis.
Acknowledgements
This work was supported, in part, by grants from the Korea Healthcare
Technology R&D Project, Ministry for Health, Welfare & Family Affairs,
Republic of Korea (A084221; to D.J.) and from the Korea Science and
Engineering Foundation (No. 2010–0001240).
Conflict of interest
The authors confirm that there are no conflicts of interest.
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Fig. S1 Flow cytometric analysis for cell surface marker expres-
sion in hASCs.
Fig. S2 Multilineage differentiation capacity of hASCs.
Fig. S3 Measurement of uterus thickness in sham-operated (Sham)
and OVX mice systemically transplanted with or without hASCs.
Fig. S4 Localization of hASCs after systemic transplantation.
Table S1 Primers used in this study
Table S2 Mass spectrometric identification of proteins secreted by
hASCs using a gel-based approach
Table S3 Mass spectrometric identification of proteins secreted by
hASCs using a non-gel based approach
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to
the corresponding author for the article.
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    • "A preclinical study of the in vivo function of human AD-MSCs by Cho et al. revealed that human AD-MSCs could prevent OVX-induced bone loss in nude mice over 8 weeks, even though there was no evidence of long-term engraftment of infused human AD-MSCs in the bone of recipient mice [137]. The effect of human AD-MSC therapy likely occurs in a paracrine manner by the secretion of various bone-related growth factors, e.g., hepatocyte growth factor, BMP-2, and RANKL, and extracellular matrix (ECM) proteins, e.g., fibronectin, which might promote osteogenic differentiation, bone remodeling and repair in the recipients [124]. Moreover, Xinhai et al. demonstrated that autologous AD-MSCs enhanced bone regeneration in OVX-induced rabbit models of osteoporosis due to not only their own osteogenic differentiation but also their promotion of osteogenesis and inhibition of adipogenesis by osteoporotic BM-MSCs through activation of BMP-2 and the BMPR-IB signaling pathway [125]. "
    [Show abstract] [Hide abstract] ABSTRACT: Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.
    Full-text · Article · Jan 2016
    • "The main advantages of ASC over MSC are a lower morbidity of the harvesting procedure and a 500-fold higher rates of precursors compared to the bone marrow biopsies when normalized to the sample volume. Moreover, an increasing body of evidence shows the ability of ASC to exert a unique paracrine (Lee et al. 2011) and immunomodulatory activity (Mariani and Facchini 2012), whose importance perhaps exceeds their ''plastic'' function based on direct lineage differentiation. With regard to cartilage regeneration, the two sources of cells present peculiar features and different culture requirements: MSC chondrogenesis requires TGF-b3, whereas ASC are more sensitive to BMP-6 (Hildner et al. 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: The first step in skeleton development is the condensation of mesenchymal precursors followed by any of two different types of ossification, depending on the type of bone segment: in intramembranous ossification, the bone is deposed directly in the mesenchymal anlagen, whereas in endochondral ossification, the bone is deposed onto a template of cartilage that is subsequently substituted by bone. Polyamines and polyamine-related enzymes have been implicated in bone development as global regulators of the transcriptional and translational activity of stem cells and pivotal transcription factors. Therefore, it is tempting to investigate their use as a tool to improve regenerative medicine strategies in orthopedics. Growing evidence in vitro suggests a role for polyamines in enhancing differentiation in both adult stem cells and differentiated chondrocytes. Adipose-derived stem cells have recently proved to be a convenient alternative to bone marrow stromal cells, due to their easy accessibility and the high frequency of stem cell precursors per volume unit. State-of-the-art "prolotherapy" approaches for skeleton regeneration include the use of adipose-derived stem cells and platelet concentrates, such as platelet-rich plasma (PRP). Besides several growth factors, PRP also contains polyamines in the micromolar range, which may also exert an anti-apoptotic effect, thus helping to explain the efficacy of PRP in enhancing osteogenesis in vitro and in vivo. On the other hand, spermidine and spermine are both able to enhance hypertrophy and terminal differentiation of chondrocytes and therefore appear to be inducers of endochondral ossification. Finally, the peculiar activity of spermidine as an inducer of autophagy suggests the possibility of exploiting its use to enhance this cytoprotective mechanism to counteract the degenerative changes underlying either the aging or degenerative diseases that affect bone or cartilage.
    Full-text · Article · Nov 2013
    • "ASC conditioned media when added to in vitro cultures of osteoclasts also led to improved survival and differentiation by ERK/JNK/p38 activation. Lee et al. analyzed the ASC secretome analysis using SDS-PAGE and mass spectrometry and detected 43 proteins of which 18 had known bone related functions [47]. "
    [Show abstract] [Hide abstract] ABSTRACT: Recent advances in protein detection and analysis have lead to multiple in depth studies that analyze the adipose-derived stem cell (ASC) secretome. These studies differ significantly in their methods of secretome preparation and analysis. Most of them use a pro-differentiation or pro-inflammatory stimulus to observe differential expression of secreted proteins. In spite of the variance in methodologies used, 68 proteins are reported to be commonly expressed in a majority of the studies and may serve as potential candidates for conserved secretome proteins. Multiple recent clinical and basic science studies demonstrate the beneficial role played by secreted proteins in augmenting ASC effects in scenarios involving angiogenesis, wound healing, tissue regeneration and immunomodulation. Furthermore, 3-D formulations of ASCs that preserve the niche environment of cells and their secreted proteins have also shown enhanced clinical effects. In light of the lack of uniformity in prior secretome-analysis studies, and the growing clinical importance of the ASC secretome, more in depth studies that use uniform and standardized means of protein detection and analysis are necessary.
    Full-text · Article · Jun 2013
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