Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells.

RESEARCH Open Access
Effects of enamel matrix derivative and
transforming growth factor-b1 on human
osteoblastic cells
Daniela B Palioto1*, Thaisângela L Rodrigues1, Julie T Marchesan1, Márcio M Beloti2, Paulo T de Oliveira2 and
Adalberto L Rosa1
Abstract
Background: Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The
objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-b1, and the
combination of both factors (EMD+TGF-b1) on human osteoblastic cell cultures.
Methods: Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of
EMD, TGF-b1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin
(OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule
formation.
Results: All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days.
At day 7, EMD group showed higher cell proliferation compared to TGF-b1, EMD + TGF-b1 and the control group.
OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were
greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP
levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group.
Conclusions: The exposure of human osteoblastic cells to EMD, TGF-b1 and the combination of factors in vitro
supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell
number, and reduced ALP activity levels and matrix mineralization.
Introduction
Periodontal regeneration is a complex series of cell and
tissue events that include cell adhesion, migration, and
extracellular matrix (ECM) protein synthesis and secre-
tion. Phenotypic expression depends on cell interactions
with ECM proteins, which regulate cell signaling events
and ultimately gene expression[1]. The ECM proteins
are, therefore, key factors that influence the regenerative
capacity[2]. However, to date, it remains undefined
which factors would determine the maximum regenera-
tive capacity.
Enamel matrix derivative (EMD) has been used in var-
ious clinical applications aiming to promote periodontal
tissue regeneration. The rationale for such application is
based on the expression of enamel matrix proteins dur-
ing the initial phases of root formation, which has been
associated with cementoblast differentiation[3,4]. In
addition, the use of EMD in various experimental and
clinical protocols has been demonstrated to positively
affect not only new cementum formation but also bone
regeneration[5-8]. However, some controversial results
in terms of new bone formation has also been described
in the literature[9].
Despite clinical evidences supporting a positive effect
of EMD on periodontal regeneration and in vitro obser-
vations on how EMD affects PDL fibroblasts[10] and
osteoblast functions[11], it is still to be clarified the
mechanisms by which EMD stimulates different period-
ontal cell types and differentiation stages. It seems to be
well determined that EMD upregulates proliferation of
* Correspondence: dpalioto@forp.usp.br
1Department of Oral Maxillofacial Surgery and Periodontology, School of
Dentistry of Ribeirão Preto - University of São Paulo, Av. do Café s/n, 14040-
904 Ribeirão Preto, SP, Brazil
Full list of author information is available at the end of the article
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HEAD & FACE MEDICINE
© 2011 Palioto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

PDL fibroblasts [10,12,13], cementoblasts[14], follicle
cells[15], and osteoblasts[16]. The controversial results
are, indeed, focused on how, and if so, EMD promotes
cell differentiation in various cell types. For instance,
while the addition of EMD in MG63 cell cultures results
in the upregulation of osteocalcin and TGF-b1[17], it
does not affect cell differentiation in other osteoblastic
cell lines[18].
Althought Gestrelius et al. [12] demonstrated that
EMD has no growth factors in its composition, others
have shown that EMD may act as a natural and efficient
drug delivery system for growth factors including TGF-
b1[19]. Additionaly, EMD can stimulate the production
of TGF-b1 by cells[17]. Indeed, PDL cells express high
levels of endogenous TGF-b1 on the presence of EMD
[20-22], raising the hypothesis that the action of EMD
would be mediated by growth factors found in its com-
position or in the culture medium modified by cells
under EMD exposure[15].
The interactions between growth factors and precur-
sor cells are key factors in the process of periodontal
healing and regeneration[23] and the association of
growth factors seems to synergistically affect the regen-
erative process[24-27]. Because the effects of the asso-
ciation of EMD with growth factors and other proteins
are still little explored, and considering that TGF-b1
regulates various cellular activities and has been
demonstrated to affect osteoblastic cell behavior, the
present study aimed to evaluate the effects of EMD,
exogenous TGF-b1 and the association of such factors
on key parameters of the development of the osteo-
genic phenotype in human alveolar bone-derived cell
cultures.
Materials and methods
Cell culture
Human alveolar bone fragments (explants) were
obtained from adult healthy donors (ranging from 15 to
25 years old), using palatal/lingual and/or interradicular
alveolar bone associated with either premolars or third
molars extracted for orthodontic reasons, with clinically
healthy periodontium. Osteoblastic cells were obtained
from these explants by enzymatic digestion using col-
lagenase type II (Gibco - Life Technologies, Grand
Island, NY) as described by Mailhot and Borke[28].
Importantly, to avoid contamination with periosteal,
periodontal ligament, and gingival cells, bone fragments
were scrapped and the first 2 digestions were discarded.
Primary cells were cultured in a-minimum essential
medium (a-MEM - Gibco), supplemented with 10%
fetal bovine serum (FBS - Gibco), 50 μg/mL gentamicin
(Gibco), 0.3 μg/mL fungizone (Gibco), 10-7 M dexa-
methasone (Sigma, St. Louis, MO), 5 μg/mL ascorbic
acid (Gibco), and 7 mM b-glycerophosphate (Sigma).
Such osteogenic culture condition supports the develop-
ment of the osteoblastic phenotype[29,30].
Subconfluent cells in primary culture were harvested
after treatment with 1 mM ethylenediamine tetraacetic
acid (EDTA - Gibco) and 0.25% trypsin (Gibco) and
subcultured cells under osteogenic culture condition
were used in all experiments. The progression of the
subcultured cells and the acquisition of the osteoblastic
phenotype have been well characterized by the work of
de Oliveira et al. [31]. During the culture period, cells
were incubated at 37°C in a humidified atmosphere of
5% CO2 and 95% air; the medium was changed every
three or four days. All experiments were performed
using three different sets of subcultures, and each
experiment conducted in quadruplicate. All patients
were informed about the study’s purpose before they
consented to participate. The local Research Ethics
Committee approved the protocol.
Treatments
Emdogain gel (EMD - Biora, Malmo, Sweden) was dis-
solved in acidic water, pH 5.9, whereas TGF-b1 (Sigma
Chemical Co., St. Louis, MO, USA) was dissolved in
acetonitrile plus trifluoracetic acid (Sigma). Both solu-
tions were aliquoted and stored at -70°C. Two concen-
trations had to be chosen because the osteoblastic cell
subculture would not allow a more extensive experi-
mental design than the one proposed herein. Thus,
based on previous studies[10,32], treatment with EMD
and TGF-b1 was performed at concentrations of 100
μg/mL and 5 ng/mL, respectively. Four experimental
conditions were established: 1) medium supplemented
with 10% FBS (control); 2) 100 μg/mL EMD in medium
supplemented with 10% FBS (EMD group); 3) 5 ng/mL
TGF-b1 in medium supplemented with 10% FBS (TGF-
b1); 4) combination of 100 μg/mL EMD and 5 ng/mL
TGF-b1 in medium supplemented with 10% FBS (EMD
+TGF-b1 group). The final pH for all groups was in the
7.2-7.4 range. A negative control was not possible
because culture medium with either no FBS or a mini-
mum concentration of FBS did not support the progres-
sion of the osteoblastic cell cultures (data not shown).
Cell growth assay
The cell growth assay was performed using a modified
method of Coletta et al. (1998)[33]. Osteoblastic cells
were plated in a 24-well culture plate (Corning Inc., NY,
USA) at a density of 20,000 cells/well in 1 mL of a-
MEM supplemented with 10% FBS (Gibco), 50 μg/mL
gentamicin (Gibco), 0.3 μg/mL fungizone (Gibco), 10-7
M dexamethasone (Sigma), 5 μg/mL ascorbic acid
(Gibco), and 7 mM b-glycerophosphate (Sigma). The
cells were allowed to attach and spread for 24 h, and
then washed with PBS and cultured in serum-free a-
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MEM for an additional 24 h. After treatments with the
four experimental conditions for four and seven days,
cells were enzymatically (1 mM EDTA, 1.3 mg/mL col-
lagenase type II, and 0.25% trypsin - Gibco) detached.
Aliquots of these solutions were incubated for 5 min
with the same volume of trypan blue and directly
counted in a hemocytometer (Fisher Scientific, Pitts-
burgh, PA, USA). For each time point, total cell number
(×104/well) was determined, which included trypan
blue-stained cells.
Bromodeoxyuridine-labeling (BrdU) index
Effect of EMD, TGF-b1 and the combination of both on
osteoblastic cells proliferation was assessed by direct
counting of cell number and BrdU incorporation into
DNA. The BrdU is detecting in the tissue through pri-
mary antibodies. These primary antibodies are then
labeled with a secondary antibody tagged with a sub-
strate for diaminobenzidine (DAB, Nunc International,
Naperville, IL, USA)[34]. The substitution of an endo-
genous DNA base, thymidine, with the BrdU analogue
ensures specific labeling of only the dividing cells during
S-phase (DNA synthesis). Osteoblastic cells were plated
on 8-well glass culture chamber slides (Nunc Interna-
tional, Naperville, IL, USA) at a density of 20,000 cells/
well in 500 μl of a-MEM supplemented with 10% FBS
(Gibco), 50 μg/mL gentamicin (Gibco), 0.3 μg/mL fungi-
zone (Gibco), 10-7 M dexamethasone (Sigma), 5 μg/mL
ascorbic acid (Gibco), and 7 mM b-glycerophosphate
(Sigma), and were incubated at 37°C and 5% CO2. Fol-
lowing 24 h of serum starvation, cells were exposed to
the four experimental culture conditions for 24 h. After
treatment, cells were incubated with BrdU (diluted
1:1,000) for 1 h under the same conditions, washed in
PBS and fixed in 70% ethanol for 15 min. BrdU incor-
poration in proliferating cells was revealed using immu-
nohistochemistry (Amershan Pharmacia Biotech Inc.,
Piscataway, NJ). Briefly, the anti-5-bromo-2’-deoxyuri-
dine monoclonal antibody, diluted 1:100 in nuclease
with deionized water, were added to the wells and incu-
bated for 1 h. The wells were then washed three times
with 500 μL of PBS and the peroxidase anti-mouse
IgG2a (15:1,000) were added to the wells and incubated
for 1 h. After another washing step, the reaction was
developed with 0.6 mg/mL of 3,3’-diaminobenzidine tet-
rahydrochloride (Sigma) containing 1% H2O2 and 1%
DMSO for 5 min at 37°C. The cells were then stained
with Crazzi hematoxylin and examined under trans-
mitted light microscopy. The BrdU labeling index,
expressed as the percentage of cells labeled with BrdU,
was determined by counting 1,500 cells using an image
analysis system (Kontron 400, Zeiss, Eching bei Munich,
Germany).
Fluorescence labeling
For immunofluorescence labeling of noncollagenous
matrix proteins, cells were treated with the four experi-
mental culture conditions for five days. At day 5, cells
were fixed for 10 min at room temperature (RT) using
4% paraformaldehyde in 0.1 M phosphate buffer (PB),
pH 7.2. After washing in PB, they were processed for
immunofluorescence labeling[31]. In addition, cell adhe-
sion and spreading were morphologically evaluated by
direct fluorescence with fluorophore-conjugated probes.
Briefly, cells were permeabilized with 0.5% Triton X-100
in PB for 10 min followed by blocking with 5% skimmed
milk in PB for 30 min. Primary monoclonal antibodies
to bone sialoprotein (anti-BSP, 1:200, WVID1-9C5,
Developmental Studies Hybridoma Bank, Iowa City, IA,
USA), alkaline phosphatase (anti-ALP, 1:100, B4-78,
Developmental Studies Hybridoma Bank), and osteopon-
tin (anti-OPN, 1:800, MPIIIB10-1, Developmental Stu-
dies Hybridoma Bank) were used, followed by a mixture
of Alexa Fluor 594 (red fluorescence)-conjugated goat
anti-mouse secondary antibody (1:200, Molecular
Probes) and Alexa Fluor 488 (green fluorescence)-conju-
gated phalloidin (1:200, Molecular Probes), which labels
actin cytoskeleton. Replacement of the primary mono-
clonal antibody with PB was used as control. All anti-
body incubations were performed in a humidified
environment for 60 min at RT. Between each incubation
step, the samples were washed three times (5 min each)
in PB. Before mounting for microscope observation,
samples were briefly washed with dH2O and cell nuclei
stained with 300 nM 4’, 6-diamidino-2-phenylindole,
dihydrochloride (DAPI, Molecular Probes) for 5 min.
After mounting with an antifade kit (Prolong, Molecular
Probes), the samples were examined under epifluores-
cence using a Leica DMLB light microscope (Leica, Ben-
sheim, Germany), with N Plan (X2.5/0.07, X10/0.25,
X20/0.40) and HCX PL Fluotar (X40/0.75, X100/1.3)
objectives, outfitted with a Leica DC 300F digital cam-
era. The acquired digital images were processed with
Adobe Photoshop software (version 7.0.1, Adobe
Systems).
Total protein synthesis
Osteoblastic cells were plated in 24-well culture plates at
a density of 20,000 cells/well in 2 mL of a-MEM sup-
plemented with 10% FBS (Gibco), 50 μg/mL gentamicin
(Gibco), 0.3 μg/mL fungizone (Gibco), 10-7 M dexa-
methasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and
7 mM b-glycerophosphate (Sigma) at 37°C in a humidi-
fied atmosphere with 5% CO2. Following serum starva-
tion, cells were exposed to the four experimental culture
conditions described previously for seven and fourteen
days. Media was changed and supplemented every three
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or four days. Total protein content was determined
using a modification of the Lowry method. Briefly, pro-
teins were extracted from each well with 0.1% sodium
lauryl sulphate (Sigma) for 30 min, resulting in a lysates
of the cells, and mixed 1:1 with Lowry solution (Sigma)
for 20 min at RT. The resulting solution was diluted in
Folin and Ciocalteau’s phenol reagent (Sigma) for 30
min at RT. Absorbance was measured at 680 nm using
a spectrophotometer (Cecil CE3021, Cambridge, UK).
The total protein content was calculated from a stan-
dard curve and expressed as μg/mL.
Alkaline phosphatase activity
Osteoblastic cells were plated in 24-well culture plates at
a density of 20,000 cells/well in 2 mL of a-MEM sup-
plemented with 10% FBS (Gibco), 50 μg/mL gentamicin
(Gibco), 0.3 μg/mL fungizone (Gibco), 10-7 M dexa-
methasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and
7 mM b-glycerophosphate (Sigma) at 37°C in a humidi-
fied atmosphere with 5% CO2. Following serum starva-
tion, cells were exposed to the four experimental culture
conditions described previously for seven and fourteen
days. Media was changed and supplemented every three
or four days. Alkaline phosphatase (ALP) was extracted
from each well with 0.1% sodium lauryl sulphate
(Sigma) for 30 min, resulting in a lysates of the cells
ALP activity was measured as the release of thy-
molphthalein from thymolphthalein monophosphate
using a commercial kit (Labtest Diagnostica, MG, Bra-
zil). Briefly, 50 μl thymolphthalein monophosphate was
mixed with 0.5 ml 0.3 M diethanolamine buffer, pH
10.1, and left for 2 min at 37°C. The solution was then
added to 50 μl of the lysates obtained from each well
for 10 min at 37°C. For color development, 2 ml 0.09 M
Na2CO3 and 0.25 M NaOH were added. After 30 min,
absorbance was measured at 590 nm and ALP activity
was calculated from a standard curve using thy-
molphthalein to give a range from 0.012 to 0.4 μmol
thymolphthalein/h/ml. Data were expressed as ALP
activity normalized for total protein content at 7 and 14
days.
Mineralized bone-like nodule formation
Osteoblastic cells were plated in 24-well culture plates at
a density of 20,000 cells/well in 2 mL of a-MEM sup-
plemented with 10% FBS (Gibco), 50 μg/mL gentamicin
(Gibco), 0.3 μg/mL fungizone (Gibco), 10-7 M dexa-
methasone (Sigma), 5 μg/mL ascorbic acid (Gibco), and
7 mM b-glycerophosphate (Sigma) at 37°C in a humidi-
fied atmosphere with 5% CO2. Following serum starva-
tion, cells were exposed to the four experimental culture
conditions described previously with differentia-
tion medium for 21 days. Media was changed and
supplemented every three or four days. At day 21, cul-
tures were washed in PBS and fixed with 10% formalde-
hyde in PBS, pH 7.2, for 16 h at 4°C. The samples were
then dehydrated in a graded series of ethanol and
stained with 2% Alizarin red S (Sigma), pH 4.2, for 8
min at RT. Using an inverted light microscope (X10
objective; Carl Zeiss, Jena, Germany), equipped with a
digital camera (Canon EOS Digital Rebel Camera, 6.3
Megapixel CMOS sensor, Canon USA Inc., Lake Suc-
cess, NY, USA), the formation of mineralized areas was
analyzed. Ten microscopic fields in each sample were
randomly selected and the mineralized area was mea-
sured as a percentage area of the well using an image
analyzer (Image Tool; University of Texas Health
Science Center, San Antonio, TX, USA).
Statistical analysis
Data represent the pooled results of three independent
experiments. Each experiment was conducted using cells
of a single donor. All experiments were performed in
quadruplicate for each set of subculture. All results are
presented as mean ± standard deviation, and the non-
parametric Kruskal-Wallis test for independent samples
was used for statistical analyses. If the result of the
Kruskal-Wallis test was significant (P <0.05), the
Fischer’s test for multiple comparisons, computed on
ranks rather than data, was performed[35].
Results
Effect of EMD, TGF-b1 or both on cell proliferation and
total cell number
Nuclear immunoreactivity for BrdU was clearly noticed
in osteoblastic cells under all treatments. Both treat-
ments and their combination affected the proliferation
at the first 24 hours of experiments compared to the
control (EMD, P < 0.001; TGF-b1, P < 0.001; EMD +
TGF-b1, P < 0.05) (Figure 1). In addition, treatment
with EMD significantly increased total cell number
compared to TGF-b1 (P < 0.05) and the combination
of the factors (P < 0.001). Treatments with EMD, TGF-
b1 and EMD+TGF-b1 significantly increased total cell
number at day 4 compared to the control (P < 0.001, P
< 0.01, and P < 0.001, respectively); the treatment with
only EMD resulted in higher values compared to the
TGF-b1 treatment (P < 0.001) and the combination of
the factors (P < 0.01), whereas total cell number for
EMD+TGF-b1 was significantly higher compared to
TGF-b1 (P < 0.01). On day 7, no statistical differences
among TGF-b1, EMD+TGF-b1 and control groups
were detected. However, all these groups showed a sig-
nificantly lower number of cells compared to the EMD
group (control, P < 0.01; TGF-b1, P < 0.05; EMD +
TGF-b1, P < 0.01) (Figure 2).
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Cellular morphology and indirect immunofluorescence for
localization of noncollagenous matrix proteins
Epifluorescence of actin cytoskeleton labeling revealed
that cells were adherent and spread, showing a polygonal
elongated morphology, with focal areas of multilayer for-
mations (Figure 3A-D). Indirect immunofluorescence
using a primary antibody anti-OPN showed that such
protein was expressed in the majority of cells, mostly in
the perinuclear area suggestive of Golgi apparatus, and in
a dot pattern throughout the cytoplasm. No differences
in terms of OPN labeling pattern and fluorescence inten-
sities among control and EMD, TGF-b1 e EMD+TGF-b1
groups were noticed; for all groups, no extracellular OPN
labeling was detected (Figure 3A-D). Immunolabeling for
ALP was more intense for control than for the treated
groups, with a labeling pattern characterized by punctate
deposits throughout the cell surface and cytoplasm
(Figure 3E-H). At day 5, no bone sialoprotein labeling
was detected for all groups (data not shown).
Effects of EMD, TGF-b1 or both on total protein synthesis,
ALP activity, and mineralized matrix formation
Total protein synthesis was not significantly affected by
the treatments (P > 0.05) (Figure 4); however, a tendency
for greater values of total protein was clearly seen at day
7 for all treated groups and for the EMD group at day
14. ALP activity was negatively affected by EMD, TGF-b1
and EMD+TGF-b1 treatments compared to the control
both at days 7 and 14. On day 14, the treatments with
EMD and EMD+TGF-b1 exhibited lower ALP activity
than TGF-b1 group (P < 0.01 and P < 0.001, respectively)
(Figure 5). At day 21, matrix mineralization was signifi-
cantly higher for the control group compared to EMD (P
< 0.05), TGF-b1 (P < 0.001) and EMD+TGF-b1 groups
(P < 0.01) (Figures 6 and 7).
Figure 1 Effect of EMD, TGF-b1 and the combination of both
factors on cell proliferation by means of BrdU-labeling at 24 h
post-treatment. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2 Effect of EMD, TGF-b1 and the combination of both
factors on cell growth. All treatments showed an increase in cell
proliferation. The EMD proliferation rate was higher than the
positive control at days 4 and 7. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3 Epifluorescence at day 5 post-treatment with the
factors. (A-D) Immunolabeling for osteopontin (OPN, red
fluorescence) was mainly cytoplasmic, in perinuclear area and in
punctate deposits. Cell-associated green fluorescence reveals actin
cytoskeleton (Alexa Fluor 488-conjugated phalloidin), whereas blue
fluorescence indicates cell nuclei (DAPI - DNA staining). No major
differences were noticed among groups in terms of labeling pattern
and fluorescence intensity for OPN. (E-H) Immunolabeling for
alkaline phosphatase (ALP, red fluorescence) was more intense for
the positive control compared to the treated groups.
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