SYMPOSIUM: TRIBUTE TO DR. MARSHALL URIST: MUSCULOSKELETAL GROWTH FACTORS
Alendronate Enhances Osteogenic Differentiation of Bone
Marrow Stromal Cells
A Preliminary Study
Hyung Keun Kim PhD, Ji Hyun Kim MS,
Azlina Amir Abbas MS (Ortho), Taek Rim Yoon MD
Received: 11 February 2008/Accepted: 8 July 2008/Published online: 30 July 2008
? The Association of Bone and Joint Surgeons 2008
However, some studies suggest alendronate also has effects
on osteoblast activity. We hypothesized alendronate would
enhance osteoblastic differentiation without causing cyto-
toxicity of the osteoblasts. We evaluated the effect of
alendronate on the osteogenic differentiation of mouse
mesenchymal stem cells. D1 cells (multipotent mouse
mesenchymal stem cells) were cultured in osteogenic dif-
ferentiation medium for 7 days and then treated with
alendronate for 2 days before being subjected to various
tests using MTT assays, Alizarin Red, enzyme-linked
immunosorbent assay, energy-dispersive xray spectropho-
tometry, reverse transcriptase–polymerase chain reaction,
confocal microscopy, and flow cytometric analysis. D1
cells differentiated into osteoblasts in the presence of
osteogenic differentiation medium as confirmed by positive
Alizarin Red S staining, increased alkaline phosphatase
activity and osteocalcin mRNA expression, a calcium peak
by energy-dispersive xray spectrophotometry, and by
Osteogenic differentiation was enhanced after treatment
with alendronate as confirmed by Alizarin Red S staining,
elevated alkaline phosphatase activity and osteocalcin
mRNA expression, a greater calcium peak by energy-
dispersive xray spectrophotometry, and by immunofluo-
rescence staining against CD44 by flow cytometric analysis.
These data suggest alendronate enhances osteogenic dif-
ferentiation when treated with mouse mesenchymal stem
cells in osteogenic differentiation medium.
Bisphosphonates are well-known inhibitors of osteoclastic
activity and are widely used to treat osteoporosis.
Alendronate is one of the most potent antiosteoporotic
agents known . The pharmacologic action of alendro-
nate relies on its interfering with the mevalonate pathway
by inhibiting farnesyl pyrophosphate synthase  and
thus reducing levels of geranylgeranyl diphosphate, which
is required for prenylation of guanosine triphosphate-
binding proteins (eg, Rab, Rac, Ras, Rho, and Cdc42) that
are essential for osteoclast activity and survival [23, 40].
Consequently, alendronate interferes with the stability of
the ruffled border and stimulates osteoclast apoptosis,
which reduces bone resorption, lowers bone turnover, and
promotes a positive bone balance .
Moreover, studies indicate bisphosphonates also influ-
ence osteoblasts [14, 19, 32, 33] and increase bone
formation [15, 20], and others have reported bisphospho-
nates enhance osteoblast proliferation and maturation [15,
20, 34] and inhibit osteoblast apoptosis . One study
exploring the effects of bisphosphonates on immortalized
human fetal osteoblasts showed decreased osteoblast
cell proliferation and increased cytodifferentiation in a
One of the authors (TRY) received funding from a Chonnam National
University (Korea) research fund.
H. K. Kim, J. H. Kim, A. A. Abbas, T. R. Yoon
Department of Orthopaedics, Chonnam National University
Hwasun Hospital, Jeonnam, Korea
H. K. Kim, J. H. Kim, T. R. Yoon
Cardiovascular Research Institute, Chonnam National
University, Gwangju, Korea
T. R. Yoon (&)
Center for Joint Disease, Chonnam National University Hwasun
Hospital, 160 Ilsimri, Hwasuneup, Hwasungun,
519-809 Jeonnam, Korea
Clin Orthop Relat Res (2009) 467:3121–3128
dose-dependent manner in cultures treated with pamidronate.
Additionally, total cellular protein, alkaline phosphatase
(ALP) activity, and Type I collagen secretion in osteoblasts
also were increased. Consistent with these findings, the rate
of bone formation also was increased in osteoblasts .
Similar findings were reported by von Knoch et al.  in
a study of the effects of bisphosphonates (alendronate,
risedronate, zoledronate) on proliferation and osteoblast
differentiation of human bone marrow stromal cells
(BMSCs). In their study using bone marrow stromal cells
from patients undergoing primary THA for end-stage
degenerative joint disease, they reported all bisphospho-
nates tested enhanced the proliferation of BMSC and
initiated osteoblastic differentiation . These observa-
tions support the suggestion that bisphosphonates have an
anabolic effect on osteoblasts and subsequently promote
bone formation, and therefore may be beneficial not only
for treatment of osteoporosis, but also for treatment of
fracture nonunion and even osteolysis.
We hypothesized alendronate would enhance osteo-
blastic differentiation compared with untreated osteoblasts
as evidenced by the presence of calcification and osteoblast
surface markers, increased ALP activity, and gene
expression. We also hypothesized this effect can be
achieved without causing cytotoxicity of the osteoblasts.
Materials and Methods
We obtained MSCs and induced osteogenic differentiation
by culturing the cells in osteogenic differentiation media
(ODM). After 3 days, the cells were divided into four
groups: Group 1 was the control group, which were cells
treated with ODM alone but not with alendronate, and
Groups 2, 3, and 4 constituted cells treated with ODM and
with 0.1, 1, or 10 lg/mL alendronate, respectively. Cells
then were tested 24 or 48 hours later to determine whether
osteogenic differentiation had been enhanced in cells
treated with alendronate compared with the control group.
The experiments included Alizarin Red S staining, ALP
activity assays, reverse transcriptase–polymerase chain
reaction (RT-PCR) analysis, scanning electron micro-
scope–energy-dispersive xray spectrometry (SEM-EDX)
analysis, immunofluorescence staining, and flow cytomet-
ric analysis. Each experiment was repeated three times for
each group, and the mean results were compared.
We cloned the primarily osteogenic D1 cells from MSCs
as described previously . D1 cells are a MSC line cloned
from Balb/c mouse bone marrow cells. These cells differ-
entiate into an osteogenic lineage when cultured in ODM,
b-glycerolphosphate. Cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum (Gibco, BRL, Bethesda, MD) and antibiotics
(Gibco). They were seeded at 1 9 104cells/cm2and
maintained in culture for 3 days in a humidified 5% CO2
atmosphere at 37?C. Experiments were performed after
cells had reached approximately 80% confluence. To
induce osteogenic differentiation, 3 days after seeding, we
changed culture media to ODM (DMEM supplemented
with 50 lg/mL ascorbic acid [Sigma-Aldrich, St Louis,
MO], 10-8mol/L dexamethasone [Sigma-Aldrich], and
10 mmol/L b-glycerolphosphate [Sigma-Aldrich]). Three
days after ODM changes, the cells were treated with 0.1, 1,
or 10 lg/mL alendronate and were analyzed 24 or 48 hours
We used fluorescence microscopy, confocal microscopy,
and flow cytometry to show changes in surface molecules,
in this case the change from MSC to osteoblast, thus
enabling identification of cells with CD44 or CD45
markers. For immunofluorescence staining, cells were fixed
with 4% paraformaldehyde in phosphate-buffered saline
(PBS) for 15 minutes, permeabilized with 0.1% Triton1
X-100 (Sigma-Aldrich, Inc., Saint Louis, MO) for
15 minutes, and then blocked with 5% bovine serum
albumen in PBS for 30 minutes. Coverslips then were
incubated with primary antibodies against mouse CD44 (an
antigen on osteoblasts, which acts as a receptor for osteo-
pontin) [20, 28] at a dilution of 1:100 and against mouse
CD45 (the leukocyte antigen) at 1:100, both at room
temperature for 1 hour. CD45 (a hematopoietic cell mar-
ker) was used as a negative control for MSCs. We then
washed cells with PBS and mounted them in 70% glycerol.
Microscopic images were obtained using an Olympus
BX50 fluorescence microscope (Olympus, Tokyo, Japan).
For flow cytometric analysis, 0.5 9 105cells were
incubated in staining buffer (PBS containing 2% fetal
bovine serum and 0.1% sodium azide) with anti-CD44,
anti-CD51, and anti-CD45 (BD Biosciences Pharmingen,
San Diego, CA) for 30 minutes in ice. We used cells that
stained with the appropriate isotype-matched immuno-
globulin as negative controls. After staining, cells were
fixed with 2% w/v paraformaldehyde and analyzed using a
FACSCaliburTMequipped with CellQuestTMsoftware (BD
Biosciences, San Jose, CA).
We assayed specific ALP activities based on the release
of p-nitrophenol from p-ntirophenyl phosphate. Optical
densities of the p-nitrophenol produced were read at
405 nm using a Multiskan1EX ELISA reader (Thermo-
Fisher Scientific, Inc, Waltham, MA). ALP activity was
normalized versus total protein content, which was deter-
mined using a QubitTMfluorometer and Quant-iTTM
protein assay kits (Invitrogen, Kingston, Ontario, Canada).
To assess the effects of alendronate on the transcriptions
of genes encoding osteocalcin (50-GAG GGC AAT AAG
GTA GTG AAC AGA-30; 50-AAG CCA TAC TGG TCT
3122 Kim et al.Clinical Orthopaedics and Related Research
GAT AGC TCG-30), osteopontin (50-CCA GGT TTC TGA
TGA ACA GTA TCC-30; 50-ACT TGA CTC ATG GCT
GCC CTT T-30), and the housekeeping enzyme glyceral-
dehyde-3-phosphate dehydrogenase (50-ATC ACT GCC
ACC CAG AAG AC-30; 50-ATG AGG TCC ACC ACC
CTG TT-30), we homogenized D1 cells grown to 70%
confluence on plates with or without alendronate using
Cincinnati, OH), and then isolated total RNA. RNA
(0.5 lg) was reverse-transcribed in 20 lL buffer contain-
ing avian myeloblastosis virus reverse transcriptase (AMV
RT) 5x, 2.5 lmol/L poly(dT), 1 mmol/L each of dATP,
dCTP, dGTP, and dTTP, 20 U RNase inhibitor, and 20 U
AMV RT. Reverse transcription was performed using the
following conditions: initial incubation at room tempera-
ture for 10 minutes and then at 42?C for 15 minutes, 99?C
for 5 minutes, and 5?C for 5 minutes in a GeneAmp1PCR
System 2700 (Applied Biosystems, Foster City, CA). Ali-
quots of cDNA were amplified in 100 lL PCR buffer
containing 15 mmol/L MgCl2, 0.1 nmol/L each of dATP,
dCTP, dGTP, and dTTP, 0.35 IU Taq DNA polymerase,
and 0.3 lmol/L 50- and 30-oligomers. PCR products were
resolved by 1.5% agarose gel electrophoresis and visual-
ized with ethidium bromide. We determined relative
quantities of amplified products using an image analysis
program (MultiGauge V3.0; Fujifilm, Tokyo, Japan).
We quantified calcification deposits in the matrices
using an assay method described by Chen et al. . Briefly,
cell cultures were washed twice with distilled water, fixed
for 1 hour in ice cold 70% (v/v) ethanol, and rinsed twice
with deionized water. Cultures were stained for 10 minutes
with Alizarin Red S, and then excess dye was removed
gently using running water. We identified calcification
deposits in the matrix, which appeared bright red, by light
microscopy and photographed. Calcification was quantified
by determining densities and areas of Alizarin Red S
staining using an image analysis program (MultiGauge
SEM-EDX analysis was performed to quantify calcium
deposits. Briefly, we treated cells with different concen-
trations of alendronate for 48 hours, and then culture dishes
were embedded in paraffin. SEM-EDX was performed
using 12-mm sections. We fixed sections to the sample
holder using a conductive carbon ribbon. A Hitachi S-4700
scanning electron microscope (Hitachi, Tokyo, Japan)
equipped with EDX was used for this work. Maps of cal-
cium distributions were acquired at 20 kV, and calcium
We performed a methyl-tetra-zolium (MTT; 3[4,5-
dimethyl-thiazoyl-2yl] 2,5-diphenyl-tetrazolium bromide;
Sigma) assay to determine the number of living cells. Cells
were treated with 0.1, 1, or 10 lg/mL alendronate and,
after 48 hours, 10 lL of MTT solution (5 mg MTT/PBS)
was added to each well and incubated for 4 hours. Finally
the 100 lL dimethylsulfoxide was added per well to sol-
ubilize the MTT-formazan. After complete solubilization
of the dye by vortexing the plate, we read absorbance on an
ELISA reader (EL 340 Biokinetics Reader; Bio-Tek
Instruments) at 570 nm wavelength. Cell viability (%) was
expressed as a ratio of alendronate-treated cells to control
MTT and ALP results are reported as mean ± standard
deviation relative to control. We used one-way ANOVA to
assess differences between treatment groups. Similarly,
gene expression is reported as mean ± standard deviation
relative to control, and one-way ANOVA was used to
assess differences. When a difference between groups was
identified by ANOVA, we compared group means using a
Student’s t test. We recorded all data in Microsoft Excel
(Microsoft, Redmond, WA), and performed all statistical
analyses using SPSS (SPSS Inc, Chicago, IL).
In the presence of ODM, all D1 cells differentiated into
osteoblasts. Cells cultured in ODM with or without
alendronate expressed CD44 (Fig. 1). However, CD45 was
not detected in any cells. Flow cytometry showed CD44
expression was increased on cells treated with alendronate,
which suggests alendronate enhanced osteoblastic differ-
entiation. Activities of ALP, a marker of early osteoblast
differentiation, were increased by 120%, 141%, and 126%
by alendronate at 0.1 (p = 0.000074), 1 (p = 0.000089),
and 10 lg/mL (p = 0.000037), respectively, compared
with cells cultured in ODM alone (Fig. 2). The mRNA
expressions of osteocalcin and osteopontin of alendronate-
treated cells also were compared with cells cultured in
ODM only (Fig. 3). Osteocalcin mRNA expression was
increased to 193%, 240%, and 272% by alendronate at
0.1 (p = 0.0054), 1 (p = 0.0044), and 10 lg/mL (p =
0.0021), respectively, versus the controls. Osteopontin
mRNA expression also was increased to 140%, 240%, and
178% by alendronate at 0.1 (p = 0.0041), 1 (p = 0.0034),
and 10 lg/mL (p = 0.0031), respectively, versus the con-
trols. Microscopic analysis and Alizarin Red S staining of
the cultured cells showed many nodules composed of
aggregated cells entrapped in a mineralized extracellular
matrix (Fig. 4). Control cells cultured in ODM alone were
stained red by Alizarin Red S, and this stain was at much
higher intensity when cells were cultured in ODM
containing 0.1, 1, or 10 lg/mL alendronate. Density mea-
surements obtained using an image analysis program
revealed cells cultured in ODM and alendronate at these
concentrations had higher intensities of 162% (p = 0.023),
Volume 467, Number 12, December 2009A Preliminary Study Using Mouse BMSC 3123
216% (p = 0.015), and 119% (p = 0.045) versus the
untreated control. SEM-EDX provided evidence of min-
eralization through the appearance of a calcium peak, and
the degree of mineralization was increased by alendronate
(Fig. 5). SEM-EDX showed no calcium peak for undif-
ferentiated D1 cells but revealed calcium peaks in cells
cultured in ODM with or without alendronate.
MTT assays at the doses of alendronate administered
showed no cytotoxicity (Fig. 6.).
Bisphosphonates are used to treat postmenopausal osteo-
porosis and are administered as prophylactics for secondary
osteoporosis. In fact, bisphosphonates are the most effec-
tive known inhibitors of bone resorption. Although they
were first used to treat Paget’s disease [26, 28], bisphos-
phonatesnow are the
hypercalcemia of malignancy and postmenopausal osteo-
porosis [17, 30]. They also were evaluated for treating
inflammation-related bone loss [1, 10], fibrous dysplasia
[7, 36], and other disorders of the musculoskeletal system,
such as osteogenesis imperfecta [2, 3]. Moreover, one
study reported bisphosphonates improve the durability of
total joint arthroplasties . We therefore hypothesized
alendronate would enhance osteoblastic differentiation
compared with ODM-treated D1 cells and that this effect
would be achieved without cytotoxicity.
As this study was only a preliminary in vitro study, we
are unsure if the findings of this experiment would be
reflected in vivo. The effects were relatively small and we
did not compare the effects of alendronate with other
known osteoblastic differentiation factors such as insulin-
like growth factor, BMP-2, and prostaglandins. Further
study is required to determine if the effects of alendronate
as seen in this study are comparable to those of these other
factors and whether they can be replicated in vitro and
The mode of action of bisphosphonates on osteoclasts
has been described [4, 11–14, 18, 32, 36, 37, 39].
Fig. 1A–C The
show increased surface molecular
expression (expression of CD44)
for MSCs cultured in ODM and
treated with alendronate by (A)
fluorescence microscopy (9100),
(B) confocal microscopy (9400),
and (C) flow cytometry.
Fig. 2 A graph shows increased ALP activity normalized protein
concentration of MSCs cultured in ODM with alendronate treatment.
Data are presented as a percentage of control (n = 3).
3124 Kim et al. Clinical Orthopaedics and Related Research
undergo apoptosis and thus reduce bone resorption, reduce
bone turnover, and promote a positive bone balance .
Bisphosphonates have well known RANKL inhibition on
fracture healing and bone strength in animal models . A
study by Mackie et al.  on expression of RANKL and
other factors in osteoclast development using pamidronate
and clodronate found a dose-related inhibition of cellular
proliferation and down-regulation of RANKL in those
cells; however, a study by Naidu et al.  showed no
effect on RANKL in response to zoledronate or alendro-
nate treatment. In addition to inhibiting bone resorption by
osteoclasts, bisphosphonates also have an anabolic effect
on osteoblasts . Mathov et al.  reported numerous
bisphosphonates (ie, olpadronate, pamidronate, etidronate)
induced rat calvaria-derived osteoblast proliferation, and
Giuliani et al.  reported bisphosphonates stimulate the
Fig. 3 Osteocalcin and osteo-
MSCs cultured in ODM and
treated with alendronate were
elevated compared with the con-
trol group. Data are presented as
a percentage of control (n = 3).
GAPDH = glyceraldehyde-3-
Fig. 4 Light microscopy photographs (original magnification, 9100)
and graphs show higher intensity of Alizarin Red S staining for MSCs
treated with alendronate. Data are presented as a percentage of control
(n = 3). ODM = osteogenic differentiation media.
Fig. 5 The diagrams show energy-dispersive xray spectrometry
analysis performed 48 hours after MSCs cultured in ODM were
treated with alendronate. The calcium peak is indicated by arrows.
The chart shows the ratio of calcium contents and percentage of
atomic weight represented by calcium of MSCs cultured in ODM and
treated with alendronate. The chart and diagrams show increases in
calcium content were in a dose-dependent manner.
Volume 467, Number 12, December 2009 A Preliminary Study Using Mouse BMSC3125
formation of osteoblast precursors and mineralized nodules
in murine and human bone marrow cultures. Im et al. 
investigated the effects of alendronate and risedronate on
primary human trabecular bone cell cultures on MG-63
osteoblast-like cell lines and found bisphosphonates pro-
moted osteoblast proliferation
evidenced by increased cell numbers and ALP activity and
by the enhanced expressions of BMP-2, Type I collagen,
and osteocalcin. Reinholz et al.  reported similar
findings in their study on pamidronate and zoledronate.
Alkaline phosphatase activity and osteocalcin and oste-
opontin expressions are considered markers of osteoblast
differentiation [6, 22], as early progenitor cells do not
express osteoblast markers such as ALP, osteocalcin, or
osteopontin  and only cells that have differentiated to a
found ALP activity and expressions of osteocalcin and
osteopontin were enhanced by alendronate in a dose-
dependent fashion. We consider it likely if alendronate
stimulates cellular differentiation, it also might stimulate
proliferation of osteocalcin and ALP-expressing cells. The
adhesion molecule CD44 is a cell surface transmembrane
glycoprotein and is encoded by a single gene. Moreover, by
acting as a receptor for hyaluronic acid, CD44 is involved in
lymphocyte activation, recirculation and homing, adhesion
to the extracellular matrix, angiogenesis and cellular pro-
liferation, differentiation, and migration . CD44
modulation also may play an important role in MSC dif-
ferentiation. Some studies suggest CD44 can be used as a
marker for osteoblasts [21, 29]. In our study, fluorescence
microscopy, confocal microscopy, and FACS analysis
showed increased presence of CD44, which mirrored the
increase in osteoblastic differentiation when cells were
treated with alendronate. Our findings suggest CD44 is a
treated with ODM and alendronate differentiate to osteo-
blasts, thus confirming alendronate can affect osteoblast
how this is achieved at the molecular level. Therefore,
additional investigations at the molecular level, involving
receptors, channels, enzymes, and signal transduction
mediators, are required to determine the mechanistic basis
of the actions of alendronate and other bisphosphonates. We
confirmed the presence of calcium in deposits by Alizarin
Red S staining and EDX. Intensities of Alizarin Red S
staining and calcium peak heights were greater in the
presence of alendronate and peaked at an alendronate con-
mineral components of calcified bone matrix, or the pre-
cursors of calcified bone matrix, were formed . Our
findings concur with those of a previous study, in which it
was suggested bisphosphonates enhanced the development
of osteoblasts from the matrix maturation stage to the
mineralization stage . Additionally, our data suggest
osteoblast cells differentiate with alendronate treatment.
Notably, we found at the highest concentration exam-
ined (10 lg/mL), alendronate had a lower effect on D1
proliferation than at 0.1 and 1 lg/mL. A similar observa-
tion was reported by Reinholz et al.  who reported an
inhibitory effect on immortalized human fetal osteoblast
cell proliferation with treatment of 10 lg/mL pamidronate
with evidence of cell death at concentrations greater than
10 lg/mL pamidronate. Im et al.  similarly observed,
at the highest concentration of alendronate used (10-4mol/
L), cellular proliferation was inhibited. In both of these
studies, it was suggested this effect was probably the result
of a toxic effect of bisphosphonates.
We found the ODM/alendronate system enhanced the
differentiation of mouse MSCs into osteoblasts in a rela-
tively dose-dependant manner. However, the real benefits
of alendronate can be established only by histologic and
clinical investigations. Moreover, our findings suggest
alendronate has a beneficial effect at the cellular level and
suggest it has clinical potential.
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