Vol.2, No.4, 133-140 (2012) Stem Cell Discovery
http://dx.doi.org/10.4236/scd.2012.24018
Increased osteogenesis with hydroxyapatite
constructs combined with serially-passaged bone
marrow-derived mesenchymal stem cells
Manabu Akahane1*, Tomoyuki Ueha2, Takamasa Shimizu2, Yusuke Inagaki2, Akira Kido2,
Tomoaki Imamura1, Kenji Kawate3, Yasuhito Tanaka2
1Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, Nara, Japan;
*Corresponding Author:
2Department of Orthopedic Surgery, Nara Medical University, Nara, Japan
3Department of Artificial Joint and Regenerative Medicine, Nara Medical University, Nara, Japan
Received 18 June 2012; revised 16 July 2012; accepted 17 August 2012
ABSTRACT
We have previously reported on both the os-
teogenic potential of hydroxyapatite (HA) com-
bined with bone marrow-derived mesenchymal
stem cells (BMSCs) and a method involving os-
teogenic matrix cell sheet transplantation of
BMSCs. In the present study, we assessed the
osteogenic potential of serially-passaged BMS-
Cs, both in vitro and in vivo. We also assessed
whether an additional cell-loading technique ca n
regain the osteogenic po tential of the constr uct s
combined with serially-passaged BMSCs. The
present study revealed that passage (P) 1 cells
cultured in osteogenic-induced medium showed
strong positive staining for alkaline phosphat-
ase (ALP) and Alizarin Red S, whereas P3 cells
showed faint staining for ALP, with no Alizarin
Red S staining. Staining of P1, P2 and P3 cells
were progressively weaker, indicating that the
osteogenic potential of the serially-passaged rat
BMSCs is lost after P3 in vitro. The in vivo study
showed that little bone formation was observed
in the HA constructs seeded with P3 cells, 4
weeks after subcutaneous implantation. How-
ever, P3 cell/HA constructs which had incre-
ased cell-loading showed abundant bone for-
mation within the pores of the HA construct.
ALP and osteocalcin mRNA expression in these
constructs was significantly higher than that of
constructs with regular cell-seeding. The pre-
sent study indicates that the osteogenic poten-
tial of the constructs with serially-passaged
BMSCs is increased by additional cell-loading.
This method can be applied to cases requiring
hard tissue reconstruction, where BMSCs require
serial expansion of cells.
Keywords: O steog enesis; Mesenchymal Stem
Cells; Serial Passaging; Hydroxyapatite; Tissue
Engineering; Bone Reconstruction
1. INTRODUCTION
Bone marrow contains a population of undifferentiated
cells known as mesenchymal stem cells (MSCs) [1,2].
Bone marrow-derived MSCs (BMSCs) have the potential
for self-renewal and differentiation into several cell types
after culture expansion in vitro and in vivo, including os-
teoblastic, chondrocytic, adipocytic and neuronal cells
[3-7]. Differentiation into osteoblastic lineage cells after
subculture in osteogenic-induced media, containing dexa-
methasone (Dex), ascorbic acid phosphate and β-gly-
cerophosphate (β-GP), has been shown for rat and human
BMSCs [8-11]. The osteogenic phenotypic markers, alka-
line phosphatase (ALP) and osteocalcin (OC), can be de-
tected by days 10 - 12 in passage 1 (P1)-cultured cells, in-
cluding mineralization [12]. The combination of BMSCs
with a scaffolding material, such as hydroxyapatite (HA),
can promote the formation of new bone tissue in vivo
after subcutaneous transplantation with freshly isolated
bone marrow cells [13] or culture-expanded BMSCs (HA/
BMSCs construct) [2,14-17], Recently, we reported a
new cell transplantation technique in which BMSCs were
cultured in culture medium containing Dex and ascorbic
acid phosphate and transplanted as cell sheets [18].
These cell sheets can be combined with HA, in which
they resulted in abundant bone formation compared with
conventional techniques [17].
Clinical applications of tissue engineered BMSCs
have been attempted in a number of therapies, including
the treatment of osteonecrosis [19-21]. In these appro-

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140
134
aches, BMSCs are obtained from the patients, following
which they generally need to be expanded by serial pass-
aging in vitro as the number of cells generated in primary
culture is limited. However, the osteogenic potential of
BMSCs might be lost by serial passaging in vitro, re-
sulting in decreased osteogenesis after subcutaneous
transplantation [8]. Therefore, it is important to deter-
mine whether HA constructs combined with serially-
passaged BMSCs maintain sufficient osteogenic potential
after in vivo transplantation. In the present study, we used
BMSCs obtained from rat bone marrow and assessed the
osteogenic potential of serially-passaged BMSCs, both in
vitro and in vivo. We also assessed whether an additional
cell-loading technique, where cells are cultured in os-
teoblastic cell sheet preparation medium can regain the
osteogenic potential of the constructs combined with se-
rially-passaged BMSCs.
2. MATERIALS AND METHODS
2.1. Bone Marrow Cell Preparation and Cell
Culture
Approval from the animal experimental review board
of Nara Medical University was obtained before begin-
ning the experiments. Institutional guidelines for the care
and use of laboratory animals have been observed. We
previously reported the method of bone marrow cell
preparation [11,18]. Briefly, bone marrow cells were ob-
tained from the femur shafts of 7-week-old male Fischer
344 rats. Both ends of the femur were removed from the
epiphysis and the marrow was flushed out using 10 ml of
standard culture medium which was expelled from a sy-
ringe containing a 20-gauge needle. Standard culture
medium consisted of minimal essential medium (MEM;
Nacalai Tesque, Kyoto, Japan) containing 15% fetal bo-
vine serum (FBS, JRH Bioscience Inc., KS, USA) and
antibiotics (100 U/ml penicillin and 100 µg/ml strepto-
mycin, Nacalai Tesque).
The obtained cells were collected in two T-75 flasks
(Falcon, BD, NJ, USA) containing 15 ml of standard
culture medium. These cells were known as passage 0
(P0). Cell culture was maintained in a humidified atom-
sphere of 95% air with 5% CO2 at 37˚C. After reaching
confluency, cells were released from the culture substra-
tum using trypsin/EDTA (Gibco, Invitrogen, CA, USA).
The released cells were seeded at density of 1 × 104
cells/cm2 for subculture in T75 flasks (Falcon) for serial
passaging to obtain passage 3 (P3) cells. Each passage of
cells (P1, P2 and P3) were used for the following ex-
periments.
2.2. ALP and Alizarin Red S Staining
To evaluate the osteogenic potential of serially-pass-
aged cells in vitro, P1, P2 and P3 cells were seeded in
6-well plates at density of 1 × 104 cells/cm2 and cultured
with or without osteogenic-induced media containing
Dex (10 nM), ascorbic acid phosphate (82 μg/mL) and
β-GP (10 mM) for 14 days. Each well was stained with
Naphtol-AS-MX phosphate sodium salt (Sigma) and Fast
Red Violet B (Nacalai Tesque) for ALP staining. 10 mg
of each reagent was dissolved in 20 ml of AMP buffer
(1.0 mM MgCl2, 10 mM p-nitrophenyl phosphate in
0.056 M 2-amino-2-methyl-propanol). Cells were incu-
bated with 2 ml of the substrate solution at room tem-
perature for ~2 minutes and rinsed with dH2O. Alizarin
Red S (0.5%) was also used to evaluate calcium deposi-
tion, following which cells were rinsed with dH2O
[12,18]. Each experiment contained 3 replicates and was
performed in duplicate.
2.3. A LP A ctivity, Calci um and DNA
Concentration in Vitro
Cells cultured in 12-well culture plates were used to
measure ALP activity, as previously reported [12].
Briefly, P1, P2 and P3 cells, seeded in 12-well plates at a
density of 1 × 104 cells/cm2 and cultured for 14 days with
or without osteogenic-induced media containing Dex,
ascorbic acid phosphate and β-GP, were scraped into 1 ml
of 0.05 M sodium phosphate buffer, 2 mM EDTA and 2
M NaCl, following which they were homogenized and
sonicated. To measure the ALP activity of cultured cells,
0.1 ml of the sonicated cell suspension was added to 0.5
ml of 0.2% Nonidet P-40 containing 1 mM MgCl2
and
centrifuged at 13,000 rpm for 10 minutes at 4˚C. The
supernatant was assayed for ALP activity using p-nitro-
phenylphosphate as a substrate. An aliquot (10 µl) of the
supernatant was added to 1 ml of 50 mM p-nitroph-
enylphosphate containing 1 mM MgCl2
and the mixture
was incubated for 30 minutes at 37˚C. Subsequently, 2 ml
of 0.2 N NaOH was added to stop the enzymatic reaction
and the absorption at 410 nm was measured by spectro-
photometry. ALP activity was represented as p-nitro-
phenol release (µmol) after 30 minutes of incubation at
37˚C.
Calcium was extracted from the sediment of Nonidet
P-40 extract with 500 µl of 20% formic acid for 24 hours
at 4˚C. An aliquot of the formic acid extract was diluted
with strontium solution. Calcium concentration was de-
termined using an atomic absorption spectrometer (A-
A-610 S; Shimadzu Co., Kyoto, Japan). The sonicated
cell suspension was also used for DNA content meas-
urement by fluorescence emission at 458 nm in the pre-
sence of 0.5 µg/ml Hoechst 33,258 (Wako Pure Chemi-
cal), with calf thymus DNA as the standard (Wako Pure
Chemical). Each group comprised of 4 wells. The experi-
ment was performed in duplicate.

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140 135
2.4. Preparation of HA Constructs Seeded
with P3 Cells
We have previously reported the preparation of HA
constructs combined with BMSCs (BMSC/HA con-
struct) [12,17]. Briefly, after release from the substratum,
P3 cells were centrifuged and resuspended at a density of
1 × 106 cells/ml in standard medium. Air was removed
from the HA, which were previously soaked in cell sus-
pension, by vacuum prior to making the constructs. Then,
the HA disks were soaked in the cell suspension for 2
hours in a CO2 incubator at 37˚C to create the P3 cell/HA
constructs. After soaking in the cell suspension, the con-
struct was transferred into a 12-well plate (Falcon) in 1
mL of osteogenic-induced medium, consisting of stan-
dard medium, 82 μg/mL ascorbic acid phosphate (Wako
Pure Chemical Industrials, Kyoto, Japan) and 10 nM Dex
(Sigma, St. Louis, MO, USA) for further subculture. After
14 days of subculture, the cultured P3 cell/HA constructs
were transplanted into a subcutaneous site of syngenic
rats for the control group of the in vivo experiments.
Porous HA ceramics (50% average void volume, 5
mm in diameter and 2 mm thickness) were used in this
study (Cellyard HA scaffold, Pentax Co., Tokyo, Japan).
The solid and porous components of the microstructure
are interconnected.
2.5. Additional Cell-Loading of the P3
Cell/HA Constructs
We previously reported that BMSCs cultured in stan-
dard medium containing Dex and ascorbic acid phos-
phate resulted in the formation of cell sheets [10,18]. In
the present study, additional P3 cells for loading were
also prepared in cell sheet culture medium. Briefly, P3
cells were seeded at a density of 1 × 104 cells/cm2 in 3.5
cm and 10 cm dishes (Falcon) and cultured in standard
medium containing Dex (10 nM) and ascorbic acid phos-
phate (82 μg/mL) (cell sheet preparation medium) until
confluent. We used a mechanical retrieval technique for
the preparation of additional P3 cells for cell loading of
the constructs. The cells were rinsed twice with phos-
phate-buffered saline (PBS; Gibco) and lifted as sheet
structure using a cell-scraper. As P3 cells formed in-
comeplete cell sheets compared with P1 cells, they were
loaded onto P3 cell/HA constructs by a short centrifuga-
tion at 900 rpm for 2 min (Kubota 5100, Kubota Corp.,
Tokyo, Japan). Subsequently, the constructs were trans-
planted into a subcutaneous site as the additional-loading
group. Because we used a number of different cell densi-
ties for the additional loading of the constructs, we
placed them into two subgroups, according to whether
they were cultured in 3.5 cm or 10 cm dishes.
Thus, there were three groups in the in vivo study: the
P3 cell/HA construct without additional loading (control
group), the P3 cell/HA construct cultured in a 3.5 cm
dish of P3 cells (small loading group) and the P3 cell/HA
construct cultured in a 10 cm dish of P3 cells (large
loading group). Each group contained seven HA disks in
two recipient rats (three or four disks per rat), which
were implanted at subcutaneous sites on their backs.
Three disks were used for histology and four for real-
time PCR analysis. The experiment was performed in
duplicate.
2.6. Sample Harvest and Histological
Analysis
Implanted disks were harvested 4 weeks after implan-
tation. HA disks for analysis by real-time PCR were fro-
zen until RNA isolation. Disks for histological evaluation
were fixed in 10% buffered formalin after trimming off
the excess rat tissue around the HA disc. The HA disks
fixed in formalin were decalcified in K-CX solution
(Falma Co., Tokyo, Japan) and embedded in paraffin.
They were cut parallel down the centre of specimens to
the round surface of the HA discs and stained with hema-
toxylin and eosin (H-E).
2.7. RNA Isolation and Real-Time
Quantitative PCR
We measured the gene expression levels of ALP and
OC to confirm osteogenesis in the harvested disks. RNA
was isolated from four disks from each group using an
Isogen RNA extraction kit (Nippon Gene Co. Ltd., To-
yama, Japan). Briefly, each sample was placed in Isogen
solution containing matrix beads and disrupted with a
FastPrep FP24 Cell Disrupter (Qbiogene, Inc., Carlsbad,
CA, USA) [12,17]. Subsequently, the remaining steps of
RNA isolation were performed according to the manu-
facturer’s instructions. To measure mRNA expression le-
vels, we conducted real-time quantitative PCR (Applied
Biosystems StepOnePlus; Applied Biosystems, Norwalk,
CT, USA), using primers for ALP, OC and glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH, internal stan-
dard) for rat cDNAs as previously described [22]. Target
ALP and OC mRNA levels were compared after correct-
ing to GAPDH mRNA levels as an internal standard, to
adjust for differences in the efficiency of reverse transcrip-
tion between samples. ALP (Rn00564931 m1), OC (Rn
01455285 g1) and GAPDH (Rn99999916 s1) primer and
probe sets were purchased from Applied Biosystems
(Foster City, CA, USA). Thermal cycle conditions were
20 sec at 95˚C for activation of TaqMan Fast Universal
PCR Master Mix, followed by 40 cycles of 1 sec at 95˚C
for denaturing and 20 sec at 60˚C for annealing and ex-
tension. This experiment was performed in duplicate.

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140
136
2.8. Statistics Figure 2 shows ALP activity and calcium deposition,
which were standardized to DNA content in each group.
The activity of P2 cells cultured in osteogenic-induced
medium were significantly lower than that of the positive
control, indicating that passaging of cells decreases the
osteoblastic phenotype of cultured cells (Figure 2(a)).
There was a significant difference in calcium deposition
between the positive control and P2 cells, indicating loss
of mineralization ability with increasing passage (Figure
2(b)).
Statistical significant was determined by one-way
ANOV
A post-hoc multiple comparisons using Tukey’s
test. A p value of 0.05 was considered statistically sig-
nificant.
3. RESULTS
3.1. Osteogenic Potential of Serial Passaged
Cells in Vitro
Figure 1 shows ALP (Figure 1(a)) and Alizarin Red
S staining (Figure 1(b)) of BMSCs that were cultured
with or without osteogenic-induced medium at P1, P2
and P3. P1 cells cultured with osteogenic-induced me-
dium showed strong positive staining for ALP and Aliza-
rin Red S, whereas P3 cells showed faint staining for
ALP and no Alizarin Red S staining. ALP staining in P2
cells seems weaker than in P1 cells and the size of mi-
neralized nodule in P2 cells appears smaller than P1 cells.
Progressively weaker staining was seen in P1, P2 and P3
cells, indicating that osteogenic potential of serially-
passaged BMSCs is lost by P3. P1, P2 and P3 cells cul-
tured without osteogenic-induced medium were negative
for ALP and Alizarin Red S staining.
3.2. Bone Ormation of P3-Cell/HA
Constructs after Implantation
Figure 3 shows representative histological sections of
the harvested HA constructs 4 weeks after implantation.
Little bone formation occurred in the pores of the control
group (the P3 cell/HA constructs: Figure 3(a)) and the
small loading group (P3 cell/HA constructs with 3.5 cm
dish P3 cell-loading: Figure 3(b)). In contrast, abundant
bone formation, including with osteoblasts and osteo-
cytes, was observed in the large loading group (P3
cell/HA constructs with 10 cm dish P3 cell-loading: Fig-
ure 3(c)).
(a) (b)
Figure 1. ALP (a) and Alizarin Red S staining (b). P1, P2 and P3 cells were cultured in 6-well
plates for 14 days. Positive staining for ALP and Alizarin Red S was observed in P1 cells cul-
tured with osteogenic-induced medium. Progressively weaker staining was seen in P1, P2 and
P3 cells cultured with osteogenic-induced medium. By contrast, P1, P2 and P3 cells cultured
without osteogenic-induced medium were negative for ALP and Alizarin Red S staining.

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140 137
(a) (b)
Figure 2. ALP activity (a) and calcium deposition (b), standardized to DNA content. P1 and P2 represent the passage of
cells. Positive and negative indicate cells cultured with or without osteogenic-induced medium, respectively. Asterisk indi-
cates p < 0.05.
(a) (b) (c)
(d) (e) (f)
Figure 3. Representative histological sections of the harvested HA constructs 4 weeks
after implantation. Little bone formation was observed in the pore of the control group
(the P3 cell/HA construct: Figures 3(a) and (b)) and small cell-loading group (the P3
cell/HA construct with 3.5 cm dish cell-loading: Figures 3(c) and (d)). In contrast,
abundant bone formation together with osteoblasts and osteocytes was seen in large
cell-loading group (the P3 cell/HA construct with 10 cm dish cell-loading: Figures 3(e)
and (f)). Arrows indicate bone tissue. Bar = 200 μm.
Figure 4 shows ALP and OC mRNA expression levels
evaluated by real-time PCR. The ALP expression in the
large loading group was significantly higher than in the
control and small loading groups (Figure 4(a)). OC ex-
pression levels were similar to ALP levels (Figure 4(b)).
4. DISCUSSION
The present study shows that serial passaging of
BMSCs decreases the osteogenic potential in vitro. Con-
sequently, little bone formation was seen in the pore of
the construct when P3 cells were combined with HA disk
(P3 cell/HA construct) and implanted into a subcutane-
ous site. However, when additional P3 cells were loaded
into the P3 cell/HA construct and implanted, obvious
bone formation was seen, indicating that the osteogenic
potential of the construct is regained by additional load-
ing of cells cultured in medium with Dex and ascorbic
acid. We assume that the additional cell-loading, using
the cell sheet culture method with short centrifugation, is
more effective at loading cells into the construct than the
conventional cell suspension method. Cell suspensions of
high cell density can be easily prepared, however, it is
difficult to achieve high loading of cells into ceramics. In
the present study, we prepare P3 cells using a 3.5 cm d

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140
138
(a) (b)
Figure 4. Real-time PCR of ALP and OC mRNA expression. OC expression in the large loading group was sig-
nificantly higher than those in the control and small loading groups (Figure 4(a)). The tendency of ALP expres-
sion (Figure 4(b)) was similar to OC expression. Control: control group, Large loading: large cell-loading group,
Small loading: small cell-loading group. Asterisk indicates p < 0.05.
and 10 cm culture dishes. The culture area of 10 cm dish
is approximately 5 times larger than the 3.5 cm dish.
Consequently, the cell density is also 5 times more in a
10 cm dish. The construct with the 3.5 cm dish P3
cell-loading showed smaller bone formation 4 weeks
after implantation compared with the 10 cm dish P3
cell-loading constructs, indicating that the loaded cell
number could be an important factor to form bone tissue
in the construct combined with serially-passaged cells.
Seeded cells may contain a combination of differen-
tiated and undifferentiated cells after passaging. Differ-
entiated cells have a limited capacity for proliferation
[23,24] and as the cells proliferating from the seeded
cells are undifferentiated cells, there is a decrease in the
percentage of osteogenic cells in the culture [8,25]. Bone
marrow cells are a mixed population of cells, including
BMSCs, fibroblasts and endothelial cells. After serial
passaging, the population of cultured cells changes. There-
fore, we assume that percentage of osteogenic cells might
decrease during cell culture and result in less ALP acti-
vity and mineralization by P2 and P3 passages.
Kumar et al. [26] have reported a method for the pre-
paration of cell sheet/HA constructs using a thermosen-
sitive polymer to fabricate the cell sheet. In their report,
they showed rapid and complete cellularization to HA by
wrapping it with a cell sheet fabricated from a human
osteosarcoma cell line. Thus, we think that it is possible
to load a lot of cells onto the construct by using the
wrapping method with P1 cell sheets. The purpose of the
present study was to evaluate the osteogenic potential of
serially-passaged cells. We used P3 cells to assess the
osteogenic potential of P3 cell/HA constructs. At P3, cell
culture with the cell sheet preparation method formed
incomplete sheet like fragments. Therefore, we used cen-
trifugation to load the incomplete cell sheet onto the
constructs as an additional cell-loading technique. Be-
cause little bone formation was seen in the harvested
construct with 3.5 cm dish cell-loading, the amount of
bone formation could depend on the loading cell number
on the HA. The decreased number of osteogenic cells
could be compensated by the additional cell-loading
from a 10 cm culture dish.
Recently, tissue engineering has been applied clini-
cally [19,20], with some of the technology using cell cul-
ture. Bone marrow cells, including BMSCs, can be ob-
tained from patients by needle aspiration and thus the
invasiveness is much less compared with harvesting the
patient’s bone. Therefore, bone marrow cells containing
BMSCs are considered a good candidate for tissue-en-
gineered bone. In general, BMSCs need to be expanded
by serial passaging to obtain enough cells for their clini-
cal application in tissue engineering. However, the os-
teogenic potential of cultured BMSCs would be lost after
serial passaging. Brugge et al. [8] reported that ALP ac-
tivity and calcium content in cultured cells were not de-
tected at P3 in vitro. They also reported that to maintain
the osteogenic potential of cells during subcultures re-
quires the addition of Dex to the culture medium as an
osteoblastic differentiation factor. However, BMSCs lose
their osteogenic potential after several passages even
when Dex was added continuously. They concluded that
the serial passaging of BMSCs results in the loss of os-
teogenic potential. A limitation of their report was that it
only contained experimental results of an in vitro study.
Therefore, we decided to examine the osteogenic poten-
tial of serially-passaged BMSCs and whether they can
differentiate into osteoblasts and subsequently promote
bone formation after in vivo transplantation. We evalu-
ated the osteogenic potential of P1-P3 cells in this study
as lower-passage BMSCs are generally used in clinical

M. Akahane et al. / Stem Cell Discovery 2 (2012) 133-140 139
applications for tissue-engineered bone reconstruction.
There are a number of limitations of the study that
should be acknowledged. First is the use of rat BMSCs in
the present study. For the clinical application of this
technique, human BMSCs should be used to determine
whether our cell-loading method can restore the oseteo-
genic potential of HA constructs combined with seri-
ally-passaged cells. Second, the culture technique for the
preparation of cell sheets formed incomplete sheets at P3.
Therefore, further improvement of cell sheet preparation
for serially-passaged cells is needed. A complete cell
sheet created by serial passaging could load large amounts
of cells onto the constructs without centrifugation and
resulted in increased bone formation. Third, we used only
HA in the present study. Other ceramics, such as β-tri-
calcium phosphate (β-TCP), are also commonly used cli-
nically [27-29]. Therefore, other types of ceramics should
be tested to confirm our results.
5. CONCLUSION
The present study indicated that osteogenic potential
of BMSCs decreases by serial passaging. However, bone
formation could be regained using the additional cell-
loading technique. Owing to its usage of serially-pas-
saged cells, this method could be applied in cases of hard
tissue reconstruction, which requires the serial passaging
of BMSCs to obtain a suitable number of cells. This
technique, along with the cell suspension method, can be
used to prepare cell-loaded constructs from ceramics,
such as HA and β-TCP.
6. ACKNOWLEDGEMENTS
We thank Ms M. Yoshimura and Ms M. Matsumura (Nara Medical
University School of Medicine, Japan) for their technical assistance.
This study was supported by Takeda Science Foundation.
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