Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid,and b-glycerophosphate may not enhance osteogenic differentiationof human periodontal ligament cells

RASHI KHANNA-JAIN1, HIDEKI AGATA1, ANNUKKA VUORINEN1,

GEORGE K. B. SANDOR1,2,3,4, RIITTA SUURONEN1,5,6, & SUSANNA MIETTINEN1

1Regea Institute for Regenerative Medicine, University of Tampere and Tampere University Hospital, Biokatu 12, 33520

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)

AbstractThis study was designed to investigate thepotential meritsof the combined useofbonemorphogeneticprotein (BMP)-2 orBMP-6and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and b-glycerophosphate] on osteogenic differentiation ofperiodontal 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-relatedtranscription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6 þ AA increased ALPactivity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPsinduced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitromineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that theaddition 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, Mesenchymalstem cells

Introduction

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

cells (PDLCs) are considered to contain hetero-

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

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

DOI: 10.3109/08977194.2010.495719

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

Growth Factors, December 2010; 28(6): 437–446

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a 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;

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

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 50mM 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.

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Cell culture with OS (dexamethasone, AA, and b-

glycerophosphate) in combination with BMP-2 or BMP-6

To investigate the synergistic effect of the combined

use of OS [10 nM dexamethasone (Sigma), 10 mM b-

glycerophosphate (Sigma) and AA] and BMP-2 or

BMP-6 on PDLCs, cells were plated in 24-well plates

at a density of 1 £ 104 cells/well, respectively, and

incubated for 24 h in B-medium. Thereafter, cells

were cultured in B-medium containing OS with or

without 10 ng/ml BMP-2 or 0.1 ng/ml BMP-6 for the

next 21 days. Culture medium was replaced with fresh

medium every 3–4 days. As a control, cells cultured

without these additives were also prepared.

Measurement of cell proliferation and ALP activity

After 7, 14, and 21 days of cell culture, cell proliferation

and ALP activity were analyzed with a commercially

available p-nitrophenyl phosphate tablet set (Sigma) as

described elsewhere (Agata et al. 2008) and cell

proliferation kit (Premix WST-1 Cell Proliferation

Assay System; Takara Bio, Inc., Shiga, Japan), with

modifications. Cell numbers (WST-1 absorbance) were

analyzed according to the manufacturer’s protocol.

Briefly, WST-1 reagents were added to each well

containing fresh medium (50ml of WST-1/500ml of

medium in each well of 24-well plate), incubated for

60 min and the absorbance was measured at 450 nm

using a microplate reader (Victor 1420, Turku, Fin-

land). After the WST-1 analysis, each well was washed

twice with PBS and p-nitrophenyl phosphate solution

was added (400ml/well for 24-well plates). After 10 min

of incubation at 378C, conversion of p-nitrophenyl

phosphate into p-nitrophenol by cellular ALP was

stopped with the equivalent amount of 3 N NaOH and

the absorbance of p-nitrophenol was measured at

450 nm using a microplate reader. ALP-specific activity

is expressed as p-nitrophenol absorbance (OD;

405 nm)/WST-1 absorbance (OD; 450 nm), which is

designed to assess the ALP activity/viable cells.

Real-time quantitative polymerase chain reaction

After 7, 14, and 21 days of culture in 6-well plates,

total RNA was extracted with Euro Gold (Euroclone

S.p.A, Pero, Italy). First-strand cDNA syntheses were

performed by a High Capacity cDNA Archive Kit

(Applied Biosystems, Warrington, UK). Real-time

quantitative PCR (qPCR) was conducted using the

primers of OC, OPN, Col I, RUNX2, and human

acidic ribosomal phosphoprotein (RPLP0) (Table I).

In order to exclude signals from contaminating DNA,

the forward and reverse sequences of each primer were

designed on different exons. The Power SYBR Green

PCR Master Mix (Applied Biosystems) was used for

qPCRs according to the manufacturer’s instructions.

The reactions were performed with Abi Prism 7300

Sequence Detection System (Applied Biosystems) at

958C 10 min, and then 40 cycles at 958C/15 s and

608C/60 s. To analyze the relative expression of OC,

OPN, Col I, and RUNX2, the Ct value of each gene

was normalized to that of the housekeeping gene

RPLP0, as described elsewhere (Pfaffl 2001).

Mineralization assays (Alizarin red S staining and

calcium deposition assay)

After 21 days of cell culture in 24-well plates, in vitro

mineralization was analyzed by alizarin red staining

and quantitative calcium assay. For Alizarin red S

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

60 min at 2208C. Then, cells were washed twice with

distilled water and stained with 40 mM Alizarin red S

solution (Sigma) for 10 min at room temperature. The

pH value of the solution was adjusted to 4.2 with 25%

ammonium hydroxide prior to staining. After staining,

excess dye was washed with distilled water and digital

images of mineral deposits were recorded.

For quantitative calcium assay, cells were washed

twice with PBS and decalcified with 0.5 N HCl

(1 ml/well) overnight at 48C. After centrifugating at

12,000 rcf for 3 min, calcium content of the

supernatant was estimated relative to a standard

provided within the Stanbio Total Calcium LiquiCo-

lor kit (Stanbio Laboratories, Boerne, TX, USA), as

described previously (Jing et al. 2007). The reaction

between calcium and ortho-cresolphthalein complex-

one produced a purple color, which was measured at

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

Potential merits of BMP-2 or BMP-6 and OS 439

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Statistical analysis

The statistical analyses of the results were performed

with GraphPad Prism 5.01. The data are presented as

mean ^ standard error of the mean (SEM) for all

quantitative experiments and represent three indepen-

dent experiments performed on cells derived from

three different donors. All statistical analyses were

performed at the significance level P , 0.05. One-way

analysis of variance (ANOVA) with Dunnett’s post hoc

test for multiple comparisons was used for the analysis.

Results

ALP activity (days 7, 14, and 21)

To investigate the effects of BMP-2 or BMP-6 on ALP

activity of PDLCs, first dose–response experiments

were conducted at 7 days time point. Figure 1 shows

dose–response results with various concentrations

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

7 days. When PDLCs were cultured with 1, 10, and

100 ng/ml concentrations of BMP-2 or BMP-6 for

7 days, 10 ng/ml of BMP-2 and 1 ng/ml of BMP-6

induced (P , 0.001) ALP activities (Figure 1(a)).

Subsequently, more detailed optimal concentration

analyses revealed that 10 or 25 ng/ml of BMP-2

induced (P , 0.001) greater ALP activity

(Figure 1(b)). In contrast, the greatest ALP activity

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

decided to use 10 ng/ml concentration of BMP-2 and

0.1 ng/ml concentration of BMP-6 for the subsequent

experiments.

Time-course experiments of cell culture with

BMP-2 þ AA or BMP-6 þ AA showed that ALP

activity was increased from days 7 to 14 (P , 0.001),

and then decreased from days 14 to 21 (Figure 2(a)).

Indu

ction

(–)

1ng/m

L BM

P-2

10ng

/mL

BMP-2

100ng

/mL

BMP-2

1ng/

mL

BMP-6

10ng

/mL

BMP-6

100ng

/mL

BMP-6

0

1

2

3

4

5

****** ***

***

ALP

act

ivity

afte

r st

anda

rdiz

atio

nA

Indu

ction

(–)

10ng

/mL

BMP-2

25ng

/mL

BMP-2

50ng

/mL

BMP-2

0

1

2

3

*** *****

ALP

act

ivity

afte

r st

anda

rdiz

atio

n

Indu

ction

(–)

0.01

ng/m

L BM

P-6

0.1ng

/mL

BMP-6

1ng/m

L BM

P-6

0

1

2

3

4

*** ******

ALP

act

ivity

afte

r st

anda

rdiz

atio

n

B C

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.

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In contrast, when BMP-2 or BMP-6 was combined

with OS, ALP activity of PDLCs continued to

increase from days 7 to 21. Although the greatest

ALP activity was observed in cells cultured with

OS þ BMP-2, there were no significant differences in

ALP activity among cells cultured with OS,

OS þ BMP-2, and OS þ BMP-6 after 21 days

(Figure 2(a)).

Cell proliferation (WST-1 values)

Cells cultured with BMP-2 þ AA or BMP-6 þ AA

showed relatively greater cell numbers than control

(induction negative control) on day 7, though

(P , 0.001) a decrease in cell number was observed

on day 21 (Figure 2(b)). In contrast, cells cultured

with OS, OS þ BMP-2, and OS þ BMP-6 showed

(P , 0.001) lower cell numbers on day 7, though

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

treated PDLCs showed a decrease in cell numbers

than control on day 21.

Cell morphology (days 14 and 21)

Representative phase contrast micrographs of PDLCs

exposed to various conditions of osteogenic induction,

after 14 days cells cultured in BMP-2 þ AA

or BMP-6 þ AA appeared as more fibroblastic and

spindle shaped (Figure 3). In contrast, cells cultured

with OS, OS þ BMP-2, or OS þ BMP-6 were rela-

tively polygonal in shape, and they started to mineralize

in vitro (Figure 3). After 21 days of culture, cells

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 (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).

Quantitative RT-PCR (days 7, 14, and 21)

The expression pattern of four osteogenic markers was

determined in the PDLCs derived from the same

further tested three donors. Time-course-related gene

expressions were investigated in PDLCs exposed to

various conditions of osteogenic induction. The time-

course results are presented at 7, 14, and 21 days.

Expression of RUNX2 mRNA

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

observed when compared with induction negative

control. Thereafter, relative RUNX2 expression

was down-regulated on days 14 and 21, compared

with the induction negative control (Figure 5(a)).

0

5

10

15

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

***************

***

******

ALP

act

ivity

afte

r st

anda

rdiz

atio

n

0.0

0.5

1.0

1.5

2.0

Induction (–) BMP-2 + AA BMP-6 + AA

OS + BMP-2 OS + BMP-6OS

*********

******

(WS

T-1

) C

ell p

rolif

erat

ion

AB

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-2þAA 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, OSþBMP-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-2þAA, 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

error bar represents the mean ^ SEM.

Potential merits of BMP-2 or BMP-6 and OS 441

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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

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

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

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).

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).

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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

0

1

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3

4

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

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

Rel

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unx2

0.0

0.5

1.0

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Induction (–) BMP-2+AA BMP-6+AA

OS OS+BMP-2 OS+BMP-6

*****

**

**

Rel

ativ

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of c

olla

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type

I

0

1

2

3

4

5

Rel

ativ

e ex

pres

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of o

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A B

C D

0

20

40

60

80

100 *

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of o

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in

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.

Potential merits of BMP-2 or BMP-6 and OS 443

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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

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

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.

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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.

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

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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