Mol Cell Toxicol (2013) 9:327-334
DOI 10.1007/s13273-013-0041-0
ORIGINAL PAPER
Effect of dental implant materials on the extracellular matrix
formation and cellular adhesion in MG-63 cells
Yu-Ri Kim1,*, Sang-Hui Seo1,*, Eun Jeong Lee1, Mi Jung Pyo1, Hye Won Kim1, Sung Ha Park1,
Dong-Woon Lee3,4, Jae-Eun Pie5, Jae-Jun Ryu3 & Meyoung-Kon Kim1,2
Received: 14 August 2013 / Accepted: 15 October 2013
�The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2013
Abstract Surface characteristics of various metallic
implant materials has been known that influence the
growth and healing in the implant-transplanted tissues.
In recent years, the use of metallic implant materials
was increased for the functional and structural recov-
ery in damaged tissues. The aim of this study is to
examine the effect of the dental implant material on
cell proliferation and attachment. In addition, we have
examined the mechanism of cell adhesion and extra-
cellular matrix (ECM) formation of implant materi-
als. The MG-63 cells were cultured on three different
implant materials such as titanium (Ti), hydroxyapatite
coated titanium (HA) and zirconia (Zr). We found that
cell proliferation on the rough surface of HA and Zr
was higher than on the smooth surface. Also, mRNA
and protein expression of ECM-related molecules,
including collagen-1, elastin and bone morphogenic
protein (BMP) was higher in cells cultured on HA
and Zr than in those on Ti. Further, phosphorylation
of Smad3 and p38 MAPK was increased in cultured
cells on HA and Zr. Thus, our data suggest that an
important determinant of cell growth and adhesion is
the suorographi thographicrface morphology of the
implant material.
1
Department of Biochemistry & Molecular Biology
2
Korean Research Institute of Molecular Medicine and Nutrition
3
Department of Dentistry, Korea University Medical School &
College, Seoul 135-705, Korea
4
Department of Periodontology, Veterans Health Service Medical
Center, Seoul 134-791, Korea
5
Department of Food and Nutrition, College of Science and
Engineering, Anyang University, Anyang 430-714, Korea
*These authors contributed equally to this work.
Correspondence and requests for materials should be addressed to
J.-J. Ryu (koprosth@unitel.co.kr),
M. K. Kim (jerrykim@korea.ac.kr)
Keywords Metal implant, Extracellular matrix, Cell
adhesion, Hydroxyapatite-coated titanium, Zirconia
Titanium is one of the biological materials that are most
commonly used and it is used for the first time as im-
plant materials late 1930s1. As titanium has great fea-
tures for implant applications such as mechano proper-
ty and lightness, it is also ideal for use in dental implant
alloy instead of chtomium and cobalt or stainless steel.
However, the metal containing more than one element
has complex properties of strength and corrosion resis-
tance, titanium alloys use more frequently than the pure
metal2-4. Hydroxyapatite (HA) is a form of calcium
phosphate and is a mineral component of bones and
teeth5,6. Because hard tissues uch as bone, dental ena-
mel and dentin contain the HA, HA is commonly used
a biomaterial due to its good mechanical properties and
excellent biocompatibility7-9. Zirconia (Zr) is ceramic
compound used for the fabrication implants10,11. In gen-
eral, Zr is hughly biocompatible and can be used for
large implants such as femoral head and total hip joint
replacemen12. Zr is also ideal for femoral head implants
due to its low friction properties and excellent biocom-
patibility11-14.
The biological process of overall development of
the interface in the dental implant is divided into two
phases. Phase I is the clinical healing phase and phase
II is the functional phase. Biological processes such
as first protein deposition, cell adhesion, cell migration
and development of ECM are generated in the healing
phase. It becomes extablishment of ECM, which is the
initial response, and leads to cellular interface with
dental implant15-22. One of the most important biologi-
cal events in clinical healing phase is a cell adhesion
at the interface between the host tissue and the dental
328 Mol Cell Toxicol (2013) 9:327-334

Table 1. Sequences of PCR primers used in this study.

Annealing
Gene Sequence Length (bp) Cycle
Tm. (�C)
F: 5′-CTACATCGACAAGGTGCGCT-3′
VIM 259 35 55
R: 5′-GGTGTTTTCGGCTTCCTCTC-3′
F: 5′-CCTCCTGGGCTGAGAGGTAG-3′
COL1A2 225 35 55
R: 5′-GCCGACAGGACCTTCTTTTC-3′
F: 5′-AGACTGTCATTGATGGGCGA-3′
FBN1 285 25 57
R: 5′-AGATCCTTCCTGTGGCATCC-3′
F: 5′-TGGTGGAGGGTATGCTCAGA-3′
ELN 256 25 55
R: 5′-CTCTAGGGCAGGCTGTGTCA-3′
F: 5′-CGGAGTCAACGGATTTGGTC-3′
GAPDH 430 25 57
R: 5′-AGCCTTCTCCATGGTGGTGA-3′

implant. This development of the ECM is related to

cell adhesion, determining characteristics of the tissue
is known. Also, ECM is an important factor in the cell
behavior, which is also crucial factor of cell prolifera-
tion. The ECM consists of structural proteins including
collagen, elastin and reticulin and fibronectin, special-
ized proteins including fibrillin and laminun, and pro-
teoblicans23-29.
Transforming growth factor-beta (TGF-β), a multi-
functional cytokine, is a regulator of ECM assembly
and remodeling. It is known that chemically and phar-
macologically stimualted healing of bone fractures and
augmented bone deposition occur via a TGF-β mediat-
ed pathway hat includes te activation of bone morpho-
genic protein (BMP). In particular, TGF-β activiety Figure 1. Cell proliferation of MG-63 cells on metal implant
inreases the expression of ECM-related molecules such materials. cells were cultured on Ti, HA or Zr for 11 days. Cell
as collagen, fibronectin, and elastin24,30,31. In addition, proliferation was assessed by MTT assay. ***P⁄0.001 HA
previous studies have reported hat TGF-β-induced syn- vs Ti, ###P⁄0.001 Zr vs Ti (Two-way ANOVA).
thesis of ECM proteins was mediated by activation of
the Smad family protein and p38 MAPK25,26.
In this study, we examined the proliferation and cell day by MTT assay. The proliferation of MG-63 cells
adhesion of human osteoblast-like MG-63 cells on the cultured on Ti, HA, or Zr steadily increased in a time-
Zr and HA-coated Ti compared to those grown on the dependent manner until 11 days. After day 5, MG-63
pure Ti. In addition, we explored how the expression cells cultured on on HA or Zr statistically grew faster
of ECM-related molecules such as collagen, elastin than that cultured on Ti (Figure 1). We can suggest that
and fibrillin were related to cell attachment to dental osteoblast cell proliferation appeared to be affected
implant materials. Finally, we investigated the activa- the disk surface used.
tion of the p38 MAPK signaling pathways and Smad
to elucidate mechanisms by expression of ECM-relat- Cell morphology cultured on different mental
ed molecules induced by TGF-β. implant materials
The surface morphology of Ti, HA and Zr and the cell
morphology of these three implant materials were
Results observed by SEM at day 7. As shown in Table 2, sur-
faces of HA and Zr were determined to be rougher
Cell proliferation assay of different metal implant
than Ti. In addition, we observed that cells cultured on
materials
HA or Zr more extended pseudopodia across the sur-
MG-63 cells were cultured on Ti, HA, or Zr for 11 face of the implant materials than that cultured on Ti.
days, and cell proliferation was measured every odd These results demonstrated that the rough morphology
Mol Cell Toxicol (2013) 9:327-334
Table 2. The appearances of surface of dental implant materials and the cell morphology.
Materials name Surface
Titanium (Ti)
Hydroxyapatite (HA)
Zrconia (Zr)
Surface of materials and cell morphology were assessed by scanning electron microscopy (SEM).
Magnification 3000X
of HA and Zr provides and ideal surface for the attach-
ment of human osteoblast cell.
Differential effects of metal implant materials on
mRNA expression of ECM structural proteins
To determine the effect of surface roughness of metal
implant materials on mRNA expression of ECM struc-
tural proteins including fibrillin-1 (FBN), elastin (ELN),
collagen-1 (COL1A2) and vimentin (VIM), we perform-
ed RT-PCR. We found that the mRNA expression of
all four ECM structural proteins was increased with
significance in cells cultured on HA or Zr, compared
to cells cultured on Ti (Figure 2). For example, the
mRNA expression of FBN1 and ELN in cells cultured
329
MG-63
in HA was increased by -2.2 and -2.1 fold (P⁄0.01
vs Ti), respectively. In addition FBN1 and ELN mRNA
levels in cells cultured on Zr were increased by -1.9
and -2.1 fold (P⁄0.01 vs Ti), respectively (Figure 2).
Differential effects of metal implant materials on the
protein expression of ECM structural proteins
To analyze the protein expression levels of ECM struc-
tural proteins including elastin and collagen-1 and
BMP1 in cells cultured on three metal implant mate-
rials, we performed Western blot analyses. Compared
to cells cultured on Ti, the expression levels of ECM
structural proteins were highly increased when cultur-
ed on HA or Zr (Figure 3). For example, the expres-
330 Mol Cell Toxicol (2013) 9:327-334

Figure 2. mRNA expression of ELN, FBN1, VIM and COL1A2 in MG-63 cells cultured on Ti, HA or Zr. MG-63 cells cultured
on Ti, HA or Zr for 7 day, after than total RNA were extracted and subjected to RT-PCR. The mRNA levels were normalized by
the basis of GAPDH expression. (A) A representative ethidium bromide (EtBr)-stained agarose gel showing ELN, FBN1, VIM
and COL1A2 mRNA levels. (B), (C), (D), (E) Quantitative analysis of FBN1, ELN, COL1A2 and VIM mRNA expression in the
gel shown in (A), respectively. *, ** indicates statistically significant differences, *P⁄0.05, **P⁄0.01 (Two-way ANOVA).

sion of elastin and BMP1 on Zr was increased by -2.7
and -2.8 fold, respectively, as compared to cells cul-
tured on Ti (Figure 3). Also, the BMP1 on HA was
increased by -2.0 fold, compared to cells on Ti (Fig-
ure 3). These results are consistent with those of our
RT-PCR analyses of ECM structural protein expres-
sion in cells grown on HA or Zr.
Activation of TGF-β signaling on metal implant
materials
It is known that TGF-β-induced ECM-related mole-
cules are mediated by the activation of Smad and p38
MAPK signaling pathways. As shown in Figure 4, the
level of phosphorylated Smad3 was elevated in cells
cultured on HA or Zr compared to those cultured on
Ti, while the amount of non-phosphorylated Smad3
was not changed. Phosphorylated Smad3 is known to
bind Smad4, and the heteromeric complex then trans-
lates into the nucleus. We found that levels of the nu-
clear form of Smad4 in cells treated on HA or Zr were
higher than those grown on Ti, whereas the level of its
cytosolic (unphosphorylated) form was not changed
in either of the cell lines on any of the three implant
Mol Cell Toxicol (2013) 9:327-334
Figure 3. Western blot analysis of BMP1, elastin, collagen-1
in MG-63 cells cultured on metal implant materials. Cells were
cultured on Ti, HA or Zr for 7 days. Equal amounts of protein
extract (25 μg) were separated on a SDS-PAGE gel, transferr-
ed to an NC membrane and incubated with BMP1, elastin, col-
lagen-1, and tubulin antibodies.
Figure 4. Western blot analysis of p38 and SMAD in MG-63
cells cultured on metal implant materials. Cells were cultured
on Ti, HA or Zr for 7 days. Equal amounts of protein extract
(25 μg) were separated on a SDS-PAGE gel, transferred to an
NC membrane and incubated with Smad3, Smad4, p38, and
tubulin antibodies.
materials (Figure 4).
Similar to Smad3, the amount of the phosphorylated
p38 MAPK was increased in MG-63 cells on HA or
Zr more than in those on Ti. In comparison to cells on
Ti, protein levels of phosphorylated p38 MAPK in
cells grown in Zr were increased by -2.3 and -1.8
fold (P⁄0.01), respectively. However, there were no
differences in the protein levels of unphosphorylated
p38 MAPK in cells on Ti, HA, or Zr (Figure 4). These
observations suggest that the increase in expression
of ECM-related molecules in cells cultured on HA or
Zr may be induced by activation of Smad3 and p38
MAPK signaling pathways that are downstream of
TGF-β.
331
Discussion
In this study, we analyzed the effect of three different
implant materials on cell adhesion and ECM formation,
including Ti which is a commonly used implant mate-
rial, hydroxyapatite-coated Ti and Zr which is a cera-
mic.
We examined the formation of ECM in cells cultured
on each of the three types of implant materials and cell
adhesion. To investigate the effect of surface morphol-
ogy of the implant material on cell proliferation and
biological response, we observed the MG-63 cells cul-
tured on Ti, HA or Zr over a 11 days. The result of
SEM showed that HA and Zr have rougher surfaces
than Ti. Since HA is fabricated by coating Ti with hy-
droxyapatite, and Zr is obtained by sandblasting and
surface treatment, they have roughness surfaces. Cell
proliferation assay showed that proliferation on HA
or Zr was significantly higher than on Ti after 5 days.
Especially, at Day 7, the proliferation in cells cultured
on HA or Zr was highly increased between about 6 to
10 folds as compared to cells on Ti. Similar to these
results, our previous studies reported that MG-63 cells
treated with HA showed higher relative increased fold
(over about 7.5 fold) at Day 6 and 7, compared to Ti
control32,33. Furthermore, osteoblast-like cells attached
to the rough surface of Zr was significantly higher than
the number of cells on the smooth surface of pure Ti13.
Some studies concluded that roughened substrate of
implant materials generated the inferior soft tissue cell
attachment34-36. Also, modified Ti surface with laser
micro-groove was shown to effective property for con-
nective tissue attachment37. These results indicate that
the ideal surface for the attachment of osteoblasts is
rougher surface such as HA and Zr.
We found that the protein levels of collagen-1 and
elastinin cells cultured on HA or Zr were higher than
those on Ti. Protein levels of BMP1 in cells grown on
HA or Zr was also higher as compared with those
grown on the Ti. It is well known that BMP is involv-
ed in the migration of progenitor cells, proliferation
and cytogenesis of mesenchymal cells, reconstruction
of bone, and vascular invasion. These results were sim-
ilar to previous studies showing the increased expres-
sion of BMP7 and COL 1, 2, and 3 in fetal osteoblast
hFOB1.19 cultured on Zr13,38. Our results revealed that
the protein levels of the structure of ECM proteins in
cells cultured on HA or Zr were higher than those on
Ti. In relation to cell attachment to the surface of den-
tal implant materials, it is reported that the expression
of ECM-related molecules were induced by TGF-β1-
Smad signaling pathway. In addition to Smad-depen-
dent signaling, the p38-MAPK pathway is triggered
by TGF-β, and p38 MAPK has been shown to increase
332
collagen expression through TGF-β mediated signal-
ing24,39. Therefore, we subsequently examined the acti-
vation levels of Smad and p38 MAPK both of which
are known to induce their expression. There was no
significant difference in total protein levels of Smad3
on Ti, HA and Zr, but the phosphorylated form of
Smad3, activated form of Smad3, was higher on HA
or Zr than on Ti. Moreover, compared to Ti control,
the phosphorylated form of p38 MAPK on HA or Zr
was increased, which indicated that the p38 MAPK
was activated in cells cultured on HA or Zr. Similarly,
it was shown that collagen synthesis induced by TGF-
β-1 was mediated by activation of p38 MAPK in Myo-
blast L6E9 cells13,30. Also, some studies reported that
Smad/p38 MAPK pathway induced the expression of
collagen26,30,40. In short, our observation and those of
others suggest that increased expression of ECM relat-
ed molecules is mediated by increased activity of p38
MAPK and Smad signaling which is stimulated by
the TGF-β.
In this study, we found that HA and Zr were able to
be an ideal surface for attachment and proliferation of
osteoblasts on dental implants. In addition, the ECM
molecules played an important role in cell attachment
to the surface of the dental implants and the increased
expression of ECM molecules was regulated by TGF-
β-1 signaling in osteoblast cells. In conclusion, our
observations provide useful information for develop-
ing and improving the surface of dental implant mate-
rials compared to other previous studies focused pri-
marily on the measurement of physical toxicity.
Materials & Methods
Dental implant materials
Three types of dental implant mateials were used in
this study: Pure titanium (Ti), hydroxyapatite coated-
titanium (HA), and zirconia (Zr). The materials were
shaped into discs measuring 10 mm in diameter and 2
mm in thickmess (OSSTEM IMPLANT CO., Ltd.).
The dental implant materials were washed in distilled
water and rinsed thoroughly in 70% absolute ethanol
before use.
Cell culture
The human osteoblast like MG-63 cells was cultured
at 37�C in a 5% CO2 humidified incubator and main-
tained in minimum essential medium (MEM), supple-
mented with 10% heat-inactivated fetal bovine serum,
2% L-glutamine and 2% penicillin and streptomycin
solution.
Mol Cell Toxicol (2013) 9:327-334
Cell proliferation assay
All titanium substrates were placed in 24-multi well
plate (Dorning, NY, USA). After completion of the
treatment, 20 μL of 5 mg/mL MTT (3-[4,5-dimethyth-
iazol-2-yl]-2,5-diphenyl-tetrazolium bromide) solu-
tion was added to the each well and incubated for 4 h
at 37� C. To dissolve the insoluble purple formazan
crystal formed, medium was replaced by dimethyl sul-
foxide (DMSO). The absorbance in each well was then
recorded at 540 nm through an ELISA reader.
Morphological analysis by scanning electron
microscopy (SEM)
The scanning electron microscope (SEM) (S-4700,
HITACHI, Tokyo, Japan) was employed in order to
determine the curvature of the dental implant materials
and morphological characteristics of cells. In this study,
MG-63 cells were cultured on Ti, Zr, or Ti for 3 days,
then the morphology of these cells were assessed by
SEM. In order to observe the appearance of dental im-
plant materials and MG-63 cells, several nanometers
of electron beam should be projected onto the object.
And then, secondary electrons are generated. Detected
electrons make light and shade. The curvature of the
surface is imaged by light and shade.
Semi-quantitative reverse transcription (RT)-PCR
analysis
Total RNA was extracted from cell using TRIzol rea-
gent (Invitrogen Co.) according to the manufacturer’s
protocol. Aliquots of 5 μg of total RNA were used to
synthesize the first-strand complementary DNA with
MMLV RTase (Invitrogen Co.) and subjected to poly-
merase chain reaction (PCR) amplification with the
oligonucleotide primers. PCR was performed using a
Taq Red DNA Polymerase (GenDEPOT. CA. USA)
system and 20 μL reaction mixes containing 2 μL
cDNA template, 2 μL 10 X reaction buffer, 0.1 mM of
dNTP, 2 μL each primer, and 2.5 U Taq polymerase.
Briefly, the samples were subjected to 10 min of dena-
turation at 95�C followed by 25-35 cycles of 30 sec at
94� C, 30 sec at annealing temperature of each primer,
and 45 sec at 72� C, and one cycle of 15 min at 72� C.
After PCR, the reaction of amplified products was re-
solved on 1.5% agarose gel and visualized with ethid-
ium bromide. PCR array sequence (Table 1).
Western blot analysis
The cells cultured on metal implant materials were
lysed with lysis buffer (GeneDEPOT, Inc), and centri-
fuged at 13,000 rpm for 30 min at 4� C. The protein
concentration in the supernatants was determined using
Mol Cell Toxicol (2013) 9:327-334
a BCA Protein Assay Kit (Sigma Aldrich, USA) and
an equal amount of extracted protein samples were
separated by 10% SDS-PAGE gels and transferred to
0.45 μm nitrocellulose (NC) membranes. Proteins were
electrophoretically transferred onto nitrocellulose trans-
fer membranes (Whatman Schelicher & Schuell, Das-
sel, Germany) in 1X transfer buffer using a Semi-dry
transfer system (Bio-rad, CA, USA). An initial block-
ing step was performed for 2 hours at room tempera-
ture with Tris-Buffered Saline Tween-20 (TBST) buf-
fer (920 mL 3rd distilled water, 30 mL 5 M NaCl, 50
mL pH 7.6 Tris-HCl, 0.5 mL Tween 20) containing
skimmed milk powder (5%) (Becton Dickinson, MD,
USA). The membranes were then rinsed to twice for
10 min each in TBST and then incubated overnight at
4� C with specific antibodies against BMP 1, elastin
and collagen 1 diluted 1 : 200. Detection was perform-
ed by using a horseradish peroxidase-conjugated rab-
bit-anti-goat secondary antibody or a horseradish pero-
xidase-conjugated goat-anti-rabbit secondary antibody
diluted 1 : 2000, and then enhanced by using Western
Blotting Luminol Reagent (Santa Cruz Biotechnology,
Inc.).
Statistical analysis
All data are expressed as the means±standard devia-
tion. After ANOVA, Duncan’s multiple range test was
performed to determine the significance of the differ-
ences in the means of cell numbers and expression
levels among cells cultured on different titanium sur-
faces. The differences were considered significant at
the 5% level.
Acknowledgements This study was supported by Rese-
arch-Driven Hospital Project of Korea University Anam
Hospital Accredited by Korean Government of Ministry
of Health and Welfare 2013.
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