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Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid, and β-

glycerophosphate may not enhance osteogenic differentiation of human
periodontal ligament cells

Article  in  Growth factors (Chur, Switzerland) · December 2010

DOI: 10.3109/08977194.2010.495719 · Source: PubMed

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 Growth Factors, 2010; Early Online: 1–10

Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid,

and b-glycerophosphate may not enhance osteogenic differentiation
of human periodontal ligament cells

RASHI KHANNA-JAIN1, HIDEKI AGATA1, ANNUKKA VUORINEN1,

GEORGE K. B. SÁNDOR1,2,3,4, RIITTA SUURONEN1,5,6, & SUSANNA MIETTINEN1
1
Regea Institute for Regenerative Medicine, University of Tampere and Tampere University Hospital, Biokatu 12, 33520
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10

Tampere, Finland, 2Regea Institute for Regenerative Medicine, University of Tampere, Tampere, Finland, 3Oral and
Maxillofacial Surgery, University of Toronto, Toronto, Canada, 4Oral and Maxillofacial Surgery, University of Oulu, Oulu,
Finland, 5Department of Eye, Ear and Oral Diseases, Tampere University Hospital, Tampere, Finland, and 6Department of
Biomedical Engineering, Tampere University of Technology, Tampere, Finland

(Received 5 March 2010; revised 18 May 2010; accepted 20 May 2010)

Abstract
For personal use only.

This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6
and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and b-glycerophosphate] on osteogenic differentiation of
periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay,
alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related
transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6 þ AA increased ALP
activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs
induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro
mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the
addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.

Keywords: Human periodontal ligament cells, bone morphogenetic protein-2 and -6, dexamethasone, ascorbic acid,
b-glycerophosphate
Abbreviations: PDLCs, Periodontal ligament cells; BMPs, bone morphogenetic proteins; ALP, alkaline phosphatase;
OPN, osteopontin; OC, osteocalcin; RUNX2, runt-related transcription factor-2; Col I, collagen type I; MSCs, Mesenchymal
stem cells

Introduction cells (PDLCs) are considered to contain hetero-

The periodontal ligament (PDL) is a soft tissue, which
connects the cementum on the roots of teeth to the
inner wall of the alveolar bone socket (Bartold et al.
2000). The main role of this connective tissue is to
maintain the teeth within the jaw, but it also plays a key
role in providing nutrition and sensation to the teeth
(Shimono et al. 2003). Thus, the periodontal ligament
geneous cell populations such as fibroblasts, neurons,
osteoblasts, cementoblasts, and undifferentiated
stem cells (Gould et al. 1980; Isaka et al. 2001).
Although the dominant cell population in PDLCs
seems to be the collagen-forming fibroblasts, several
studies suggest that PDLCs have osteoblasts-like
properties, because PDLCs show high levels
of alkaline phosphatase (ALP) activity and a

Correspondence: R. Khanna-Jain, Regea Institute for Regenerative Medicine, University of Tampere, 33520 Tampere, Finland.
Tel: 358 4 1901789. Fax: 358 3 35518498. E-mail: rashi.khanna-jain@regea.fi

ISSN 0897-7194 print/ISSN 1029-2292 online q 2010 Informa UK Ltd.

DOI: 10.3109/08977194.2010.495719
 2 R. Khanna-Jain et al.
capacity to form mineralized nodules after
osteogenic induction (Murakami et al. 2003; Hayami
et al. 2007). The PDLCs are also known to express the
osteoblasts-associated genes such as runt-related
transcription factor-2 (RUNX2), collagen type I
(Col I), osteopontin (OPN), and osteocalcin (OC)
(Saito et al. 2002; Inanc et al. 2006). However, the
time-course expressions of these genes in PDLCs
during osteogenic differentiation have not been
delineated.
Bone morphogenetic proteins (BMPs), originally
identified as proteins that induce bone formation at
extra-skeletal sites, are multifunctional growth factors
that regulate the growth, differentiation, and apoptosis
of various cell types, including osteoblasts, chondro-
blasts, neural cells, and epithelial cells (Urist 1965;
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Hogan 1996). Currently, there are 14 subsets of
BMPs which belong to the transforming growth
factor-b superfamily. Four of them (BMP-2, -4, -6,
and -7) are known as inducers of osteogenic
differentiation (Lavery et al. 2008). BMP-6 has been
reported to be one of the potent inducers of osteogenic
differentiation in mesenchymal stem cells (MSCs;
Friedman et al. 2006). As the information concerning
For personal use only.
the effect of BMP-6 on hPDLCs is still limited (Xu
et al. 2004), it is important to investigate their cellular
and molecular characteristics of hPDLCs cultured in
the presence of BMP-6.
Dexamethasone, ascorbic acid (AA), and b-
glycerophosphate (osteogenic supplements: OS) are
the most widely used supplements to induce the
osteogenic differentiation of various species-derived
stem/progenitor cells including human MSCs and
PDLCs (Nohutcu et al. 1997; Hayami et al. 2007).
Interestingly, a recent study suggested that simul-
taneous induction with both BMP-2 and OS
accelerated the osteogenic differentiation of human
MSCs (Jager et al. 2008), whereas some studies
reported that the induction with BMP-2 alone fails to
induce osteogenic differentiation of human MSCs
(Osyczka et al. 2004; In Sook et al. 2008; Mizuno
et al. 2009). Regarding hPDLCs, the results of
osteogenic induction with BMP-2 alone are incon-
sistent (Kobayashi et al. 1999; Hou et al. 2007),
though the effect of simultaneous induction with
both BMP-2 and OS remains to be investigated.
With respect to the combined induction with BMP-6
and OS, there is far less information available than
with BMP-2 and OS.
In the present study, we aimed to investigate the
cellular and molecular characteristics of hPDLCs
cultured in the presence of BMP-2 or BMP-6.
Furthermore, the combined effect of OS and BMP-2
or BMP-6 were also investigated.
Materials and methods
Cell isolation and culture
This study was conducted with the approval of the
Ethics Committee of the Pirkanmaa hospital district,
Tampere, Finland (R06009). Human-impacted third
molars were obtained from seven patients aged 21–26
years (23 ^ 2.5 years) with informed consent at the
Finnish Student Health Services, Tampere, Finland.
The teeth samples were brought from the health center
to the laboratory in phosphate buffered saline (PBS;
BioWhittaker Lonza, Verviers, Belgium) containing 2%
antibiotics/antimycotics (100 U/ml penicillin,
0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin
B; Invitrogen, Paisley, Scotland, UK) and PDL tissue
fragments were harvested from the middle third of the
roots of teeth. Following tissue extraction, the PDL
tissue fragments were digested with collagenase type I
3 mg/ml (Invitrogen) and dispase 4 mg/ml (Invitrogen)
for 1 h at 378C. Once digestion was completed, cell
suspension was obtained by passing through a 70-mm
strainer (Falcon, BD Labware, Franklin lakes, NJ,
USA) and cells were expanded in 75 cm2 culture flasks
(Nunc, Roskilde, Denmark) in basic cell culture media
(B-medium) consisting of Dulbecco’s modified Eagle’s
medium (DMEM/F-12 1:1; Invitrogen), 10% fetal
bovine serum (Invitrogen), L -glutamine (GlutaMAX I;
Invitrogen), and 1% antibiotics/antimycotics. Cells
cultured in 75 cm2 flasks were incubated at 378C in
5% CO2 and the medium was changed 2–3 times per
week. When PDLCs reached 80% confluence, PDLCs
were detached with trypsin solution (BioWhittaker
Lonza), and divided into new flasks. The harvested cells
were cryopreserved and stored in liquid nitrogen until
used for the experiments. To avoid potential artifacts of
passage number on cellular phenotype or their
molecular expression, PDLCs at passages 3 and 4
were used in the present study.
Cell culture with BMP-2 or BMP-6
To investigate the effect of BMP-2 or BMP-6 on
PDLCs, cells were plated in 24-well plates (Nunc) at a
density of 1 £ 104 cells/well, respectively, and were
incubated for 24 h in B-medium. Thereafter, cells were
cultured in B-medium containing various concen-
trations of recombinant human BMP-2 (1, 10, 25, 50,
and 100 ng/ml; Sigma, St Louis, MO, USA) or BMP-6
(0.01, 0.1, 1, 10, and 100 ng/ml; R&D Systems,
Hameenlinna, Finland) and 50 mM L -ascorbic acid 2-
phosphate (AA; Sigma) for the next 7 days. To
investigate the effect of longer duration of culture with
each BMP, cells in B-medium containing AA and
10 ng/ml BMP-2 or 0.1 ng/ml BMP-6 were also
prepared and cultured for the following 21 days. Culture
medium was replaced with fresh medium every
3–4 days. Control cells were cultured without these
additives.
 Potential merits of BMP-2 or BMP-6 and OS 3

Cell culture with OS (dexamethasone, AA, and b- S.p.A, Pero, Italy). First-strand cDNA syntheses were
glycerophosphate) in combination with BMP-2 or BMP-6 performed by a High Capacity cDNA Archive Kit
(Applied Biosystems, Warrington, UK). Real-time
To investigate the synergistic effect of the combined
quantitative PCR (qPCR) was conducted using the
use of OS [10 nM dexamethasone (Sigma), 10 mM b-
primers of OC, OPN, Col I, RUNX2, and human
glycerophosphate (Sigma) and AA] and BMP-2 or
acidic ribosomal phosphoprotein (RPLP0) (Table I).
BMP-6 on PDLCs, cells were plated in 24-well plates
In order to exclude signals from contaminating DNA,
at a density of 1 £ 104 cells/well, respectively, and
the forward and reverse sequences of each primer were
incubated for 24 h in B-medium. Thereafter, cells
designed on different exons. The Power SYBR Green
were cultured in B-medium containing OS with or
PCR Master Mix (Applied Biosystems) was used for
without 10 ng/ml BMP-2 or 0.1 ng/ml BMP-6 for the
qPCRs according to the manufacturer’s instructions.
next 21 days. Culture medium was replaced with fresh
The reactions were performed with Abi Prism 7300
medium every 3– 4 days. As a control, cells cultured
Sequence Detection System (Applied Biosystems) at
without these additives were also prepared.
958C 10 min, and then 40 cycles at 958C/15 s and
608C/60 s. To analyze the relative expression of OC,
Measurement of cell proliferation and ALP activity
OPN, Col I, and RUNX2, the Ct value of each gene
After 7, 14, and 21 days of cell culture, cell proliferation was normalized to that of the housekeeping gene
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10

and ALP activity were analyzed with a commercially RPLP0, as described elsewhere (Pfaffl 2001).
available p-nitrophenyl phosphate tablet set (Sigma) as
described elsewhere (Agata et al. 2008) and cell
proliferation kit (Premix WST-1 Cell Proliferation Mineralization assays (Alizarin red S staining and
Assay System; Takara Bio, Inc., Shiga, Japan), with calcium deposition assay)
modifications. Cell numbers (WST-1 absorbance) were
After 21 days of cell culture in 24-well plates, in vitro
analyzed according to the manufacturer’s protocol.
mineralization was analyzed by alizarin red staining
Briefly, WST-1 reagents were added to each well
and quantitative calcium assay. For Alizarin red S
containing fresh medium (50 ml of WST-1/500 ml of
For personal use only.

staining, cells were fixed with ice-cold 70% ethanol for

medium in each well of 24-well plate), incubated for
60 min at 2 208C. Then, cells were washed twice with
60 min and the absorbance was measured at 450 nm
distilled water and stained with 40 mM Alizarin red S
using a microplate reader (Victor 1420, Turku, Fin-
solution (Sigma) for 10 min at room temperature. The
land). After the WST-1 analysis, each well was washed
pH value of the solution was adjusted to 4.2 with 25%
twice with PBS and p-nitrophenyl phosphate solution
ammonium hydroxide prior to staining. After staining,
was added (400 ml/well for 24-well plates). After 10 min
excess dye was washed with distilled water and digital
of incubation at 378C, conversion of p-nitrophenyl
images of mineral deposits were recorded.
phosphate into p-nitrophenol by cellular ALP was
For quantitative calcium assay, cells were washed
stopped with the equivalent amount of 3 N NaOH and
twice with PBS and decalcified with 0.5 N HCl
the absorbance of p-nitrophenol was measured at
(1 ml/well) overnight at 48C. After centrifugating at
450 nm using a microplate reader. ALP-specific activity
12,000 rcf for 3 min, calcium content of the
is expressed as p-nitrophenol absorbance (OD;
supernatant was estimated relative to a standard
405 nm)/WST-1 absorbance (OD; 450 nm), which is
provided within the Stanbio Total Calcium LiquiCo-
designed to assess the ALP activity/viable cells.
lor kit (Stanbio Laboratories, Boerne, TX, USA), as
described previously (Jing et al. 2007). The reaction
Real-time quantitative polymerase chain reaction
between calcium and ortho-cresolphthalein complex-
After 7, 14, and 21 days of culture in 6-well plates, one produced a purple color, which was measured at
total RNA was extracted with Euro Gold (Euroclone 544 nm using a microplate reader.

Table I. Primer sequences for quantitative RT-PCR.

Name 50 -Sequence-30 Product size Accession number

RPLPO Forward 50 -AAT CTC CAG GGG CAC CAT T-30 70 NM_001002
Reverse 50 -CGC TGG CTC CCA CTT TGT-30
OC Forward 50 -AGC AAA GGT GCA GCC TTT GT-30 63 NM_000711
Reverse 50 -GCG CCT GGG TCT CTT CAC T-30
OPN Forward 50 -GCC GAC CAA GGA AAA CTC ACT-30 71 J04765
Reverse 50 -GGC ACA GGT GAT GCC TAG GA-30
Col I Forward 50 -CCA GAA GAA CTG GTA CAT CAG CAA-30 94 NM_000088
Reverse 50 -CGC CAT ACT CGA ACT GGA ATC-30
RUNX2 Forward 50 -CCCGTGGCCTTCAAGGT-30 76 NM_004348
Reverse 50 -CGTTACCCGCCATGACAGTA-30
 4 R. Khanna-Jain et al.

Statistical analysis of BMP-2 or BMP-6 in combination with AA at

7 days. When PDLCs were cultured with 1, 10, and
The statistical analyses of the results were performed
with GraphPad Prism 5.01. The data are presented as 100 ng/ml concentrations of BMP-2 or BMP-6 for
mean ^ standard error of the mean (SEM) for all 7 days, 10 ng/ml of BMP-2 and 1 ng/ml of BMP-6
quantitative experiments and represent three indepen- induced (P , 0.001) ALP activities (Figure 1(a)).
dent experiments performed on cells derived from Subsequently, more detailed optimal concentration
three different donors. All statistical analyses were analyses revealed that 10 or 25 ng/ml of BMP-2
performed at the significance level P , 0.05. One-way induced (P , 0.001) greater ALP activity
analysis of variance (ANOVA) with Dunnett’s post hoc (Figure 1(b)). In contrast, the greatest ALP activity
test for multiple comparisons was used for the analysis. was observed at 0.1 ng/ml for BMP-6 (Figure 1(c)).
As there was no significant difference in ALP activity
between 10 and 25 ng/ml BMP-2 (Figure 1(b)), we
Results decided to use 10 ng/ml concentration of BMP-2 and
0.1 ng/ml concentration of BMP-6 for the subsequent
ALP activity (days 7, 14, and 21) experiments.
To investigate the effects of BMP-2 or BMP-6 on ALP Time-course experiments of cell culture with
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10

activity of PDLCs, first dose –response experiments BMP-2 þ AA or BMP-6 þ AA showed that ALP
were conducted at 7 days time point. Figure 1 shows activity was increased from days 7 to 14 (P , 0.001),
dose – response results with various concentrations and then decreased from days 14 to 21 (Figure 2(a)).

A
5
ALP activity after standardization

4 ***
***
*** ***
For personal use only.

0
)

6
(–

P-

P-

P-

P-

P-

P-
n

BM

BM

BM

BM

BM

BM
io
ct

L
du

/m

/m

m
In

g/

g/

g/

g/
ng

ng
1n

0n

1n

0n
10

10
10

10

B C
3 4
ALP activity after standardization

ALP activity after standardization

***
*** 3 *** ***
** ***
2

1
1

0 0
)

6
)
(–

P-

P-

P-

P-

P-

P-
(–
n

BM

BM

BM

BM

BM

BM
n
tio

io
uc

ct
L

L
/m

/m

/m

du

/m

m
d
In

g/

g/
ng

ng

ng

In

ng

1n

1n
10

25

50

01

0.
0.

Figure 1. Cell culture with various concentrations of BMP-2 or BMP-6. (A) hPDLCs were cultured with AA and 1, 10, and 100 ng/ml of
BMP-2 or BMP-6 for 7 days. Among these concentrations, 10 ng/ml of BMP-2 and 1 ng/ml of BMP-6 induced relatively greater ALP
activities. (B) When PDLCs were cultured with 10, 25, and 50 ng/ml of BMP-2, relatively greater ALP activity was observed in 10 and
25 ng/ml. (C) When PDLCs were cultured with 0.01, 0.1, and 1 ng/ml of BMP-6, relatively greater ALP activity was observed in 0.1 ng/ml.
ALP activity of each treatment was standardized by that of induction (2). Error bar represents the mean ^ SEM. Significant differences from
control, ***P , 0.001 and **P , 0.01.
 Potential merits of BMP-2 or BMP-6 and OS 5

A
15 B
2.0

ALP activity after standardization

(WST-1) Cell proliferation

*** 1.5
10 ***
***
*********
*** *** 1.0
***
5 ****** *** ***
0.5

0 0.0
Day 7 Day 14 Day 21 Day 7 Day 14 Day 21

Induction (–) BMP-2 + AA BMP-6 + AA

OS OS + BMP-2 OS + BMP-6
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Figure 2. Cell culture with BMP-2, BMP-6, OS, or in combination. hPDLCs were exposed to (1) BMP-2 (10 ng/ml) þ AA; (2) BMP-6
(0.1 ng/ml) þ AA; (3) OS (dexamethasone, AA, and b-glycerophosphate); (4) OSþBMP-2 (10 ng/ml); or (5) OSþBMP-6 (0.1 ng/ml), and
cultured for 7, 14, and 21 days to evaluate their effect on cell proliferation and ALP activities. (A) All treated groups showed significant
increase in ALP activities than induction (2) on day 14. ALP activities of cells cultured with OS, OSþBMP-2, or OSþBMP-6 were
significantly greater than those of cells cultured with BMP-2þ AA or BMP-6þAA following the 21-day time course. ALP activity of each
treatment was standardized by that of induction (2) at day 7. Statistically significant difference from the control of day 7 was analyzed,
***P , 0.001 and error bar represents the mean ^ SEM. (B) PDLCs cultured with OS, OSþ BMP-2, or OSþBMP-6 showed a significant
decrease in cell number than cells cultured with BMP-2þAA, BMP-6þAA, or without induction (induction (2)), on day 7, though those
differences were hardly observed on day 14. On day 21, all the treated PDLCs showed smaller cell number than induction (2) whereas a
significant decrease in cell numbers were observed in cells treated with BMP-2þ AA, BMP-6þAA. WST-1 value of each treatment was
standardized by that of induction (2) at day 7. Statistically significant difference from the control of day 7 was analyzed, ***P , 0.001 and
For personal use only.

error bar represents the mean ^ SEM.

In contrast, when BMP-2 or BMP-6 was combined or BMP-6 þ AA appeared as more fibroblastic and
with OS, ALP activity of PDLCs continued to spindle shaped (Figure 3). In contrast, cells cultured
increase from days 7 to 21. Although the greatest with OS, OS þ BMP-2, or OS þ BMP-6 were rela-
ALP activity was observed in cells cultured with tively polygonal in shape, and they started to mineralize
OS þ BMP-2, there were no significant differences in in vitro (Figure 3). After 21 days of culture, cells
ALP activity among cells cultured with OS, cultured with BMP-2 þ AA or BMP-6 þ AA started to
OS þ BMP-2, and OS þ BMP-6 after 21 days detach from the bottom of the culture plate edge and
(Figure 2(a)). formed cell aggregates (Figure 4). On the other hand,
cells cultured with OS, OS þ BMP-2, or OS þ BMP-6
showed the ability to mineralize in vitro (Figure 4).
Cell proliferation (WST-1 values)
Cells cultured with BMP-2 þ AA or BMP-6 þ AA Quantitative RT-PCR (days 7, 14, and 21)
showed relatively greater cell numbers than control The expression pattern of four osteogenic markers was
(induction negative control) on day 7, though determined in the PDLCs derived from the same
(P , 0.001) a decrease in cell number was observed further tested three donors. Time-course-related gene
on day 21 (Figure 2(b)). In contrast, cells cultured expressions were investigated in PDLCs exposed to
with OS, OS þ BMP-2, and OS þ BMP-6 showed various conditions of osteogenic induction. The time-
(P , 0.001) lower cell numbers on day 7, though course results are presented at 7, 14, and 21 days.
those differences were hardly observed on day 14, and
cell number on day 21 was relatively lower than
control again (Figure 2(b)). Interestingly, all the Expression of RUNX2 mRNA
treated PDLCs showed a decrease in cell numbers
than control on day 21. When cells were cultured with BMP-2 þ AA or
BMP-6 þ AA, RUNX2 expression was up-regulated
on day 7, whereas no significant differences were
Cell morphology (days 14 and 21)
observed when compared with induction negative
Representative phase contrast micrographs of PDLCs control. Thereafter, relative RUNX2 expression
exposed to various conditions of osteogenic induction, was down-regulated on days 14 and 21, compared
after 14 days cells cultured in BMP-2 þ AA with the induction negative control (Figure 5(a)).
 6 R. Khanna-Jain et al.
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10
Figure 3. Representative phase contrast photographs of the cells
cultured with BMP-2, BMP-6, and OS (day 14). Phase contrast
micrographs of hPDLCs were exposed to (1) induction ( 2 ) without
additives as control; (2) OS (dexamethasone, AA, and
For personal use only.
b-glycerophosphate); (3) BMP-2 (10 ng/ml) þ AA; (4) OS þ BMP-
2 (10 ng/ml); (5) BMP-6 (0.1 ng/ml) þ AA; or (6) OS þ BMP-6
(0.1 ng/ml), and cultured for 14 days. Morphologically, cells cultured
with BMP-2 þ AA or BMP-6 þ AA appeared as more fibroblastic
and spindle cells in shape. Cells cultured with OS, OS þ BMP-2, or
OS þ BMP-6 were relatively polygonal in shape, and they started to
mineralize in vitro (black arrow). Original magnification ( £ 40).
Cells cultured with OS, OS þ BMP-2, or
OS þ BMP-6 also showed the RUNX2 up-regu-
lation on day 7 and down-regulation on days 14
and 21. However, no significant differences were
observed for RUNX2 expression of PDLCs
between each treatment.
Expression of COL I mRNA
As there was no increase in the Col I expression at day
7, no differences between the various groups was
observed. Cells exposed to BMP-2 þ AA, OS,
OS þ BMP-2, or OS þ BMP-6 (P , 0.001) down-
regulated Col I expression following days 7 –21 time
course (Figure 5(b)).
Expression of OPN and OC mRNA
The level of OPN mRNA expression in control cells
was fairly low in comparison to the treated cells
following the 21 days time course. The cells treated
with OS showed time-dependent (P , 0.05) increase
of OPN expression at day 21 whereas the addition of
BMP-2 or BMP-6 down-regulated OPN expression
Figure 4. Representative phase contrast photographs of the cells
cultured with BMP-2, BMP-6, and OS (day 21). Phase contrast
micrographs of hPDLCs were exposed to (1) induction (2) without
additives as control; (2) OS (dexamethasone, AA, and b-
glycerophosphate); (3) BMP-2 (10 ng/ml) þ AA; (4) OS þ BMP-2
(10 ng/ml); (5) BMP-6 (0.1 ng/ml) þ AA; or (6) OS þ BMP-6
(0.1 ng/ml), and cultured for 21 days. PDLCs cultured with BMP-
2 þ AA or BMP-6 þ AA started to detach from the bottom of the
culture plate edge and formed cell aggregates (white arrow). Cells
cultured with OS, OS þ BMP-2, or OS þ BMP-6 showed the ability
to mineralize in vitro (black arrow). Original magnification ( £ 40).
as shown in (Figure 5(c)). In contrast, OC mRNA
expression was stable at all time points and there were
no differences in OC expression between the control
and cells exposed to BMPs þ AA or OS þ BMPs
following the 21-day time course (Figure 5(d)).
Mineralization of PDL cells (day 21)
Calcium deposition assay. At 21 days, P , 0.001,
increase in calcium content of the cells cultured in
OS, OS þ BMP-2, or OS þ BMP-6 was seen, as
shown in Figure 6(a). In contrast, calcium deposition
was hardly observed in the cells exposed to BMP-
2 þ AA, BMP-6 þ AA (data not shown), and control.
There were, however, no significant differences
between the calcium content of the cells treated with
OS, OS þ BMP-2, or OS þ BMP-6.
Alizarin red staining. The biomineralization ability
of PDLCs was also analyzed by alizarin red staining
as shown in (Figure 6(b)). Cells exposed to
 Potential merits of BMP-2 or BMP-6 and OS 7

Induction (–) BMP-2+AA BMP-6+AA

OS OS+BMP-2 OS+BMP-6

A B

Relative expression of collagen type I

4 1.5
Relative expression of Runx2

3
1.0

0.5 *
*
1
**
*****
0 0.0
Day 7 Day 14 Day 21 Day 7 Day 14 Day 21
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C D
5

Relative expression of osteocalcin

Relative expression of osteopontin

100 *
4
80

3
60

40 2

1
For personal use only.

20

0 0
Day 7 Day 14 Day 21 Day 7 Day 14 Day 21

Figure 5. The expression profiles of osteoblasts-related genes. hPDLCs were exposed to (1) BMP-2 (10 ng/ml) þ AA; (2) BMP-6
(0.1 ng/ml) þ AA; (3) OS (dexamethasone, AA, and b-glycerophosphate); (4) OS þ BMP-2 (10 ng/ml); or (5) OS þ BMP-6 (0.1 ng/ml), and
cultured for 7, 14, and 21 days to evaluate their effect on the expression of osteoblasts-related genes. Cells cultured without these additives
were also prepared as a control (induction (2)). (A) Regardless of the conditions for osteogenic induction, the RUNX2 expression was
up-regulated on day 7, and it was down-regulated on days 14 and 21. (B) The expression of Col I was significantly down-regulated by day 21 in
the cells treated with OS, OS þ BMP-2, and OS þ BMP-6. Statistically significant difference from the control of day 7 was analyzed,
***P , 0.001, *P , 0.05. (C) Increased expression of OPN was observed following days 7– 21 regardless of the conditions of the osteogenic
induction used. However, significant up-regulation of OPN expression was only observed in cells cultured with OS on day 21, though large
variations were observed between donors (n ¼ 3). Statistically significant difference from the control of day 7 was analyzed, *P , 0.05. (D)
The expression of OC was not up-regulated following the time course in all of the treated groups. Error bar represents the mean ^ SEM.

BMP-2 þ AA and BMP-6 þ AA did not show matrix
mineralization of the PDLCs (data not shown).
Consistent with the results of calcium deposition
assays, there seemed no significant differences
between the intensities of alizarin red staining of the
cells treated with OS, OS þ BMP-2, and OS þ BMP-6
(Figure 6(a),(b)). Microscopic images also suggested
that treatment with OS resulted in extracellular matrix
formation and eventual mineralization in 14–18 days of
induction (Figures 3 and 4).
Discussion
BMPs are multifunctional growth factors which are
involved in the regulation of cell proliferation,
survival, differentiation, and apoptosis of various
types of cells, and their hallmark abilities are to induce
bone, cartilage, ligament, and tendon formation
(Groeneveld and Burger 2000; Xiao et al. 2007).
BMP-2 is one of the most extensively studied BMPs,
and the combined use of BMP-2 and OS has been
shown to be able to accelerate the osteogenic
differentiation of human MSCs (Jager et al. 2008).
However, as reported in a recent study (Mizuno et al.
2009), the response of human MSCs to BMP-2 is
known to vary between donors, resulting in inconsist-
ent effect of BMP-2 on in vitro osteogenic differen-
tiation of human MSCs. Moreover, clinical studies of
bone regeneration by BMP-2 have also reported
inconsistent results between patients (Groeneveld and
Burger 2000; Govender et al. 2002). Taking into
account the previous findings that the in vitro
osteogenic induction efficacy of BMP-6 is greater
than that of BMP-2 in MSCs (Friedman et al. 2006),
it is reasonable to hypothesize that the combined use
of BMP-6 and OS is more effective than that of
 8 R. Khanna-Jain et al.
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10
Figure 6. The mineralization in vitro. hPDLCs were exposed to
(1) OS (dexamethasone, AA, and b-glycerophosphate);
(2) OS þ BMP-2 (10 ng/ml); or (3) OS þ BMP-6 (0.1 ng/ml), and
cultured for 21 days to evaluate their effect on the in vitro
mineralization. Cells cultured without these additives are also
prepared as a control (induction (2)). (A) Calcium deposition assay
For personal use only.
revealed that there was no significant difference in the ability of
mineralization in vitro among the cells cultured with OS,
OS þ BMP-2, or OS þ BMP-6. The results of calcium deposition
in each treatment were analyzed for statistical significant difference
in comparison to the control (induction (2)), P , 0.001. Error bar
represents the mean ^ SEM. (B) Alizarin red S staining of the cells
cultured with OS, OS þ BMP-2, or OS þ BMP-6.
BMP-2 and OS or OS alone. Thus, we investigated
the effect of BMP-2, BMP-6, OS, OS þ BMP-2, or
OS þ BMP-6 on the osteogenic differentiation of
hPDLCs.
First, we investigated the effect of BMP-2 or BMP-
6 on the osteogenic differentiation of hPDLCs.
The results of 7- or 14-day culture with BMP-2 or
BMP-6 alone showed that both BMPs could increase
the ALP activity, though following the time-course
experiments, they revealed that neither of BMPs could
induce in vitro mineralization of PDLCs by day 21. In
contrast to these findings, a previous study reported
that BMP-2 could induce in vitro mineralization in
murine PDL cell lines (Saito et al. 2002). Although
the reasons for this discrepancy remain to be
investigated, one of the reasons might not be the
difference in dose of BMPs (see supplemental figure),
but the species difference in responsiveness to BMP-2
between murine and human cells, as suggested
elsewhere (Mizuno et al. 2009). This diversity of
BMP-responsiveness between human and rodent cells
should be further delineated. With respect to the effect
of BMP-2 or BMP-6 on cell proliferation of PDLCs,
there was a decrease in cell numbers following the
21-day time course, which is consistent with the
results reported in human MSCs and PDLCs
(Kobayashi et al. 1999; In Sook et al. 2008).
The synergistic effect of the combined use of OS
and BMP-2 in promoting osteogenic differentiation
has been reported in human MSCs (In Sook et al.
2008; Jager et al. 2008). However, the combined effect
of OS and BMP-6 or BMP-2 has not been elucidated
in hPDLCs. Our study revealed that cells cultured
with OS þ BMP-6 as well as OS þ BMP-2 showed
relatively greater ALP activity than cells cultured with
OS alone at 21 days time point, though there were no
significant differences in the in vitro mineralization
ability among them. Although seven donor-derived
cells were cultured for our experiments, evidence of
PDLCs mineralization was observed in three donor
samples only. In accordance with our findings, we
report here the variability in mineralization potential
between donors of hPDLCs.
As the cells cultured with OS þ BMP-2 or
OS þ BMP-6 showed relatively greater ALP activity
than cells cultured with OS alone, we subsequently
performed qRT-PCR analyses. We investigated
whether RUNX2, Col I, OPN, and OC mRNAs,
which are known to be expressed in PDLCs as well as
osteoblasts (Owen et al. 1990; Stein et al. 1990; Lian
and Stein 1992; Saito et al. 2002; Inanc et al. 2006),
are up-regulated by the addition of BMP-2 or BMP-6
to OS. In contrast to our expectations, cells cultured
with OS þ BMP-2 or OS þ BMP6 did not show any
greater expression of these mRNAs than cells cultured
with OS alone. Rather, cells cultured with OS alone
showed relatively greater expression of OPN. Our data
suggest that the osteogenic differentiation of hPDLCs
is sufficiently induced by OS, and that the addition of
BMP-2 or BMP-6 to OS may not produce any
significant synergistic effects.
Although BMPs are known to be powerful
osteogenic inducers and are involved in tooth
morphogenesis (Aberg et al. 1997; Xiao et al. 2007),
BMPs have failed to induce osteogenic differentiation
in rat PDL cells (Rajshankar et al. 1998). In contrast,
an in vivo study reported that BMP-6 increased bone
and cementum formation in a rat model (Huang et al.
2005). Our results, however, demonstrate that the
addition of BMP-6 to OS or BMP-6 alone did not
enhance osteogenic differentiation of PDLCs. The
reason for this discrepancy could be due to the
difference in the responsiveness to BMP-6 between
species, which needs to be further elucidated.
Considering the inconsistent response to BMPs, this
study highlights the potential merits of OS in
osteogenic differentiation of PDLCs. In fact, there
are many reports showing the osteogenic potential of
PDLCs under the influence of OS alone (Nohutcu
et al. 1997; Kuru et al. 1999; Hayami et al. 2007).
These findings are very important in terms of potential
future cost savings. Assuming that OS are shown

to be safe, the less expensive OS can be shown to be
more effective than BMPs or the combination of OS
and BMPs, in inducing osteogenic differentiation of
PDLCs. Thereby, significant costs of growth factors
could be saved both in the research setting and
potentially in the future cell therapy environments.
In conclusion, this study showed that the combined
use of OS and BMP-2 or BMP-6 does not provide an
efficient osteogenic induction protocol for hPDLCs.
These in vitro studies are important for defining the
responses of PDLCs to BMPs, and are, therefore,
necessary for guiding their clinical use. However,
these results highlight the need to further investigate
the molecular mechanism of osteogenesis in PDLCs
in response to BMPs.
Growth Factors Downloaded from informahealthcare.com by 93.106.52.234 on 06/23/10
Acknowledgments
The authors thank Ms. Anna-Maija Honkala for
her excellent technical assistance. We are indebted to
Ms. Bettina Lindroos for her constructive comments
of the manuscript.
Declaration of interest: This work was supported by
the Finnish Funding Agency for Technology and
For personal use only.
Innovation (TEKES), competitive Research Funding
of Pirkanmaa hospital district and University of
Tampere. The authors report no conflicts of interest.
The authors alone are responsible for the content and
writing of the paper.
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