African Journal of Biotechnology Vol. 8 (20), pp.

5522-5526, 19 October, 2009

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB09.509
ISSN 16845315 2009 Academic Journals

Full Length Research Paper

In vitro assessment of cytotoxicity of giomer on human

gingival fibroblasts
Reza Pourabbas1, Safar Farajnia2*, Soodabeh Kimyai3, Leila Mohammadnejad3, Antony
Johnson4 and Touraj Nejatian4
1

Drug Applied Research Center, Tabriz University, Medical Sciences, Tabriz, Iran.
Biotechnology Research Center, Tabriz University, Medical Sciences, Tabriz, Iran.
3
Faculty of Dentistry, Department of Operative Dentistry, Tabriz University, Medical Sciences, Tabriz, Iran.
4
Department of Adult Dental Care, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.
2

Accepted 15 May, 2009

Root coverage on restored root surfaces has been considered as a challenging issue. The evaluation of
cytotoxic effects of restorative materials is a fundamental requirement for sustaining the cell
attachment and the clinical success of root coverage. The aim of the present study was to compare the
human gingival fibroblast cytotoxicity of the recently introduced giomer composite (GC) with resin
ionomer (RI) restorative material. Discs (62 mm) of GC and RI restorative materials were prepared
using sterile Teflon mold. Extracts from the materials were incubated to cell culture medium for 24, 48
and 72 h. Human gingival fibroblasts (HGF) were exposed to the extracts of the materials while the unincubated media served as the control group. The cytotoxicity of the materials were evaluated by 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In order to compare the mean
values of the measured parameters a Kruskal-Walis test was carried out. MTT assay indicated that
human gingival fibroblasts proliferated well in the presence of GC extract. The proliferation rate was
higher in cells incubated with GC compared to RI extracts but the differences were not statistically
significant (p= 0.09). This in vitro study indicated that GC is a non-toxic material for HGF. However,
further studies are needed to assess the other biologic and clinical behavior of this material prior to it
being considered as a potentially suitable restorative material to restore the carious root lesions
candidated to root coverage procedures.
Key words: Root coverage material, giomer, ionomer, cell cytotoxicity.
INTRODUCTION
Gingival recession is defined as the displacement of the
gingival margin apical to the cemento-enamel junction
(Deliperi et al., 2006). In a study, gingival recession of 1
mm or more was reported in 11.5% of 18 to 24 year olds,
46.3% of 35 to 44 year olds, 78.3% of 55 to 64 year olds
and 86.5% of people 65 or older (Brown et al., 1996).
Gingival recession predisposes the affected teeth to loss
of cervical structure, dental hypersensitivity and root
caries (Seichter, 1987).
Root coverage procedures such as sub-epithelial connective tissue grafts (SCTG) are used successfully to

*Corresponding author. E-mail: farajnias@tbzmed.ac.ir. Tel:

0098411-3363234.

cover the exposed root surfaces (Langer and Langer,

1985). However, in some complicated cases where the
gingival recession and root caries are present, a combination of restorative and periodontal surgical procedures
needs to be undertaken. In such circumstances, the carious lesion is removed and the resultant cavity is restored with a proper restorative material such as resin ionomer (Geristore) before being covered by a mucoperiosteal flap and /or graft (Alkan et al., 2006). Owing to its
composition and physico-mechanical properties, Geristore can be used in treating sub-gingival defects such
as root resorption and perforation (Alkan et al., 2006;
Dragoo and Scherer 1995). Furthermore, in laboratory
studies, Geristore was less cytotoxic to gingival fibroblasts than conventional glass ionomer (Ketac-Fil) and
Immediate Restorative Materials (IRM) (Al-Sabak et al.,

Pourabbas et al.

2005).
Recently, a new fluoride releasing light cured restorative material containing pre-reacted glass ionomer fillers,
known as giomer, has been introduced (Yap and Mok.,
2002) based on the incorporation of surface pre-reacted
(S-PRG) or full pre-reacted (F-PRG) glass ionomer fillers.
In the S-PRG type, only the surface of the glass filler is
attacked by polyacrylic acid leaving the glass core intact,
while in the F-PRG type, the entire filler particle is attacked by polyacrylic acid (Sunico et al., 2005). According to
the claims made by the manufacturer, giomer combines
the advantages of a resin composite and a glass ionomer
(Deliperi et al., 2006). These new hybrid materials were
found to provide almost a complete seal against bacterial
microleakage, cause a little mechanical and chemical
pulpal irritation and inhibited demineralization (Sunico et
al., 2005; Sonoda et al., 2002; ISO 10993-12., 1996). In a
study, it was shown that the two year clinical performance of giomer restorations was equal or slightly better than
that of resin ionomers and compomers (Sunico et al.,
2005).
Due to the high success rate of giomers in cervical
restorations, it is assumed that these materials can be
used in combination with SCTG for the coverage of carious root surfaces. Since the knowledge around the cytotoxicity of giomers towards human gingival fibroblasts is
limited, this study aimed to assess the citotoxicity of giomer (Beautifil) on human gingival fibroblasts and compare it with the citotoxicity of a resin ionomer (Geristore).
METHODS AND MATERIALS
Sample preparation
6 mm diameter and 2 mm thick discs of Beautifil (Shofu Dental
Co-orporation, Osaka. Japan) and Geristore (DEN-MAT Coorporation, Santa Maria. CA. USA) were fabricated under aseptic
conditions by packing the materials in a Teflon mold and compressed between two glass plates to create a even thickness disc.
The discs were sterilized by suspending in 70% ethanol for 10 min
before the experiment.
Cell culture
Human gingival fibroblast cells were cultivated and maintained in
RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal calf serum, 100 U ml-1 penicillin, 100 lL ml-1
streptomycin and 2 mmol L-1 l-glutamine (Cambrex Bio Science,
Verviers, Belgium) at 37C in a humidified atmosphere of 95% air
and 5% CO2 as described (Farajnia et al., 2008). Sub-cultivation
was performed with cells from confl-uent cultures treated with 0.2 g
L-1 ethylenediaminetetraaceticacid in phosphate-buffered saline
(PBS).

5523

48-h-incubation periods, the test discs were transferred into the 96well plate and washed 3 times with media to remove unattached
cells. The attachment of viable cells on the surfaces of discs was
assessed using the MTT assay. A total of 20 l MTT dye 3-(4, 5dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide) (5 mg/ml
in PBS) was added to each well and the microplates were incubated at 37C for additional 2 h. After the incubation period, 100 l of
acidified isopropanol (0.04 N HCI in isopropanol) was added to the
wells and mixed thoroughly to dissolve the dark blue crystals of formazan. The absorbance values of each well were determined with
a microplate enzyme-linked immuno-assay (ELISA) reader at a
570-nm wavelength. Untreated cultures were used as control
groups and their survival rates were set to represent 100% viability.
The results were calculated as 100(absorbance of test well/absorbance of positive control well) 100 and presented as a percentage
of cell proliferation. Each experiment was repeated 3 times and the
mean value was calculated.
Preparation of discs extracts and cell cytotoxicity assay
Eight discs from each product were immersed in RPMI 1640 media
and agitated for 48 and 72 h at 37C according to ISO Standard
10993-12 (Gonzales et al., 2004). The control samples containing
only medium were treated similarly. HGF cells were diluted in a
fresh medium containing 2, 5 and 10% of FCS and seeded into 96well plates (10-4 cells well-1). After incubation for 24 h, the medium
was aspirated from all wells and replaced by 100 l well-1 extraction or control medium and incubated for another 24 h before cytotoxicity was checked. The colorimetric assay developed by
Mosmann (Mosmann, 1983) and modified by Edmondson et al.
(Edmondson et al., 1988) was used as a test for cell proliferation
and survival assay. A total of 20 l MTT dye (5 mg ml-1 in PBS)
was added to each well and incubated at 37C, in air containing 5%
CO2 and at 95% relative humidity for 4 h in the dark. After incubation, the MTT was aspirated and the formazan product was dissolved in 100 l of the acidified isopropanol (0.04 N HCI in isopropanol). The plates were shaken before the optical densities (OD)
were measured at a 570 nm wavelength. Three tests of each extract and control were performed in each experiment. All assays
were repeated at least twice to ensure reproducibility.

Statistical analysis
In order to compare the mean values of the measured parameters a
Kruskal-Walis test was carried out. Two sided values of p<0.05
were accepted as significant.

RESULTS
Attachment assay
The attachment of human gingival fibroblasts on Beautifil and Geristore based discs was assessed by MTT
assay after 24 and 48-h incubation. The results are given
in Table 1. More HGF cells were attached to Beautifil
discs than for the Geristore discs but the difference was
not statistically significant (2= 1.33; p=0.24).

Attachment assay
For analysis of cell attachment, the test discs were placed in the
center of a 24-well-cell culture plate under sterile conditions and
then the cell suspension was added (104 cells/well). After 24 and

Cell cytotoxicity assay

The cytotoxicity of the Beautifil based discs on human

Pourabbas et al.

2005).
Recently, a new fluoride releasing light cured restorative material containing pre-reacted glass ionomer fillers,
known as giomer, has been introduced (Yap and Mok.,
2002) based on the incorporation of surface pre-reacted
(S-PRG) or full pre-reacted (F-PRG) glass ionomer fillers.
In the S-PRG type, only the surface of the glass filler is
attacked by polyacrylic acid leaving the glass core intact,
while in the F-PRG type, the entire filler particle is attacked by polyacrylic acid (Sunico et al., 2005). According to
the claims made by the manufacturer, giomer combines
the advantages of a resin composite and a glass ionomer
(Deliperi et al., 2006). These new hybrid materials were
found to provide almost a complete seal against bacterial
microleakage, cause a little mechanical and chemical
pulpal irritation and inhibited demineralization (Sunico et
al., 2005; Sonoda et al., 2002; ISO 10993-12., 1996). In a
study, it was shown that the two year clinical performance of giomer restorations was equal or slightly better than
that of resin ionomers and compomers (Sunico et al.,
2005).
Due to the high success rate of giomers in cervical
restorations, it is assumed that these materials can be
used in combination with SCTG for the coverage of carious root surfaces. Since the knowledge around the cytotoxicity of giomers towards human gingival fibroblasts is
limited, this study aimed to assess the citotoxicity of giomer (Beautifil) on human gingival fibroblasts and compare it with the citotoxicity of a resin ionomer (Geristore).
METHODS AND MATERIALS
Sample preparation
6 mm diameter and 2 mm thick discs of Beautifil (Shofu Dental
Co-orporation, Osaka. Japan) and Geristore (DEN-MAT Coorporation, Santa Maria. CA. USA) were fabricated under aseptic
conditions by packing the materials in a Teflon mold and compressed between two glass plates to create a even thickness disc.
The discs were sterilized by suspending in 70% ethanol for 10 min
before the experiment.
Cell culture
Human gingival fibroblast cells were cultivated and maintained in
RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal calf serum, 100 U ml-1 penicillin, 100 lL ml-1
streptomycin and 2 mmol L-1 l-glutamine (Cambrex Bio Science,
Verviers, Belgium) at 37C in a humidified atmosphere of 95% air
and 5% CO2 as described (Farajnia et al., 2008). Sub-cultivation
was performed with cells from confl-uent cultures treated with 0.2 g
L-1 ethylenediaminetetraaceticacid in phosphate-buffered saline
(PBS).

5523

48-h-incubation periods, the test discs were transferred into the 96well plate and washed 3 times with media to remove unattached
cells. The attachment of viable cells on the surfaces of discs was
assessed using the MTT assay. A total of 20 l MTT dye 3-(4, 5dimethyl-thiazoyl)-2,5-diphenyl-SH-tetrazolium bromide) (5 mg/ml
in PBS) was added to each well and the microplates were incubated at 37C for additional 2 h. After the incubation period, 100 l of
acidified isopropanol (0.04 N HCI in isopropanol) was added to the
wells and mixed thoroughly to dissolve the dark blue crystals of formazan. The absorbance values of each well were determined with
a microplate enzyme-linked immuno-assay (ELISA) reader at a
570-nm wavelength. Untreated cultures were used as control
groups and their survival rates were set to represent 100% viability.
The results were calculated as 100(absorbance of test well/absorbance of positive control well) 100 and presented as a percentage
of cell proliferation. Each experiment was repeated 3 times and the
mean value was calculated.
Preparation of discs extracts and cell cytotoxicity assay
Eight discs from each product were immersed in RPMI 1640 media
and agitated for 48 and 72 h at 37C according to ISO Standard
10993-12 (Gonzales et al., 2004). The control samples containing
only medium were treated similarly. HGF cells were diluted in a
fresh medium containing 2, 5 and 10% of FCS and seeded into 96well plates (10-4 cells well-1). After incubation for 24 h, the medium
was aspirated from all wells and replaced by 100 l well-1 extraction or control medium and incubated for another 24 h before cytotoxicity was checked. The colorimetric assay developed by
Mosmann (Mosmann, 1983) and modified by Edmondson et al.
(Edmondson et al., 1988) was used as a test for cell proliferation
and survival assay. A total of 20 l MTT dye (5 mg ml-1 in PBS)
was added to each well and incubated at 37C, in air containing 5%
CO2 and at 95% relative humidity for 4 h in the dark. After incubation, the MTT was aspirated and the formazan product was dissolved in 100 l of the acidified isopropanol (0.04 N HCI in isopropanol). The plates were shaken before the optical densities (OD)
were measured at a 570 nm wavelength. Three tests of each extract and control were performed in each experiment. All assays
were repeated at least twice to ensure reproducibility.

Statistical analysis
In order to compare the mean values of the measured parameters a
Kruskal-Walis test was carried out. Two sided values of p<0.05
were accepted as significant.

RESULTS
Attachment assay
The attachment of human gingival fibroblasts on Beautifil and Geristore based discs was assessed by MTT
assay after 24 and 48-h incubation. The results are given
in Table 1. More HGF cells were attached to Beautifil
discs than for the Geristore discs but the difference was
not statistically significant (2= 1.33; p=0.24).

Attachment assay
For analysis of cell attachment, the test discs were placed in the
center of a 24-well-cell culture plate under sterile conditions and
then the cell suspension was added (104 cells/well). After 24 and

Cell cytotoxicity assay

The cytotoxicity of the Beautifil based discs on human

5524

Afr. J. Biotechnol.

Table 1. Assessment of the attachment of human gingival fibroblasts on Beautifil and Geristore discs.

% FCS
OD

Cells + Beautifil disc

2.5
5
10
1.17
1.47
1.62
0.21
0.12
0.32

Cells + Geristore disc

2.5
5
10
1.43
1.7
1.80
0.63
0.11
0.23

Positive control
2.5
5
10
1.06
1.39
1.62
0.41
0.47
0.50

Negative control
2.5
5
10
0.43
0.47
0.56
0.02
0.03
0.02

OD, optical density; FCS, Fetal Calf Serum.

Data are expressed in mean SD.

Table 2. Assessment of the cytotoxicity of Beautifil and Geristore based discs on human gingival fibroblasts.

OD

G 24 h
1.430.25

G 484 h
1.580.11

G 72 h
1.50.13

B 48 h
1.640.21

B 48 h
1.4016

B 24 h
1.680.31

Cell
1.550.2

Media
0.380.06

OD = Optical density, G = Geristore, B = Beautifil.

Data are expressed in mean SD.

gingival fibroblasts were assessed by proliferation analysis of cells incubated with Beautifil disc extracts using
Geristore discs extracts as a control. As shown in Table
2, Beautifil discs 24, 48 and 72 h extracts did not show
any cytotoxicty and there were no statistically significant
differences between the proliferation rate of cells cultured
in the culture medium and that of cells incubated with
Geristore and Beautifil extracts (2= 4.75; p=0.09).
DISCUSSION
Restorative materials must be biocompatible to minimize
their adverse effects on periodontal tissues induced by
direct contact (Souza et al., 2006). In vitro tests are often
preferred to measure biocompatibility aspects in the early
stages of the assessment of a newly introduced material,
in the interest of time, cost and ethics. While in vitro
studies are easier to conduct, their validity can only be
substantiated by careful and meticulous in vivo research
(Schmalz, 1994; Sidhu and Schmalz, 2001).
In the present study, the method of MTT-colorimetric
assay was used as an in vitro method to assess the
fibroblast cell survival and proliferation rate measuring
dehydrogenase activity as described in previous studies
(Mosmann, 1983; Huang et al., 2002).
Like other tissues, normal fibroblast function is critical
to maintain the periodontal tissue function for optimal
healing. Gingival fibroblasts were chosen due to their
availability and culturing characteristics (Huang et al.,
2002; Hou and Yaeger, 1993).
The results of the present study revealed that
fibroblasts attached well on the surface of the both tested
materials. In addition, there was no statistically significant
difference between cell proliferation rate of control group
(culture medium alone), resin ionomer (Geristore) and
giomer (Beautifil) extracts. Our results were in agreement with the previous study in which fibroblasts grew

and spread well over Geristore with a morphology close

to that of the control groups in SEM evaluation (Al-Sabak
et al., 2005). Moreover, in a study carried out by Camp et
al. (Camp et al., 2003) on Geristore group, fibroblast
attachment occurred significantly greater than for other
investigated root-end filling materials (Camp et al., 2003).
Based on histologic evidence, Dragoo (Dragoo, 1997)
demonstrated that both epithelium and connective tissue
can adhere to resin ionomer placed in a subgingival environment. Resin ionomers have also been used successfully in subgingival areas to treat furcation lesions and
restore resorption cavities (White, 1998; Breault et al.,
2000; Anderegg, 1998).
It is really difficult to clarify why these two materials especially giomer showed acceptable results in cytotoxicity
tests because investigators often do not know the exact
composition of materials being tested. However, there
might be some possible reasons; favorable cellular response in Geristore and Beautifil might be attributed to
their surface characteristics as the surface structure of
the final restoration can often determine the biocompatibility of the material (Yap and Mok, 2002; Dunkin and
Chambers, 1983). The amount and nature of leachable
compounds in resinous materials can influence their biocompatibility (Huang et al., 2002), as a result, less eluted
toxic substances into the medium by those restorative
materials is another possible explanation for the good cell
attachment and proliferation. In resin base materials, free
monomers and additives are extracted especially during
the first 24 h. Therefore, monomer-polymer conversion is
a very important factor in biocompatibility of resinous
materials (Ferracane and Condon, 1990).
Even though there were not any statistically significant
differences between the two materials, giomer exhibited
higher fibroblast attachment compared with the resin
ionomer. This might be due to its better polishability compared to the resin ionomer (Yap and Mok, 2002). Also, it
has been revealed that the low initial pH of dental mate-

Pourabbas et al.

rials may lead to cytotoxic reactions (Sidhu and Schmalz,

2001; Meryon et al., 1983). Since giomer employs prereacted glass ionomer technology, the fluoroalumino
silicate glass reacts with polyalkenoic acid in water prior
to the inclusion into silica-filled urethane resin (Yap and
Mok., 2002), it seems that the initial pH in giomer does
not decrease as much as that of resin ionomer. A study
demonstrated that the resin modified glass ionomers
maintained a low surface pH for at least the first 60 min of
setting (Woolford and Chadwick, 1992).
Unlike the results of Al-Sabak et al. (2005), Camp et al.
(2003) and the findings of this current study, Huang et al.
(2002) had demonstrated that resin-modified glass ionomer cement was cytotoxic to cultured human gingival
fibroblasts by inhibiting cell growth, attachment and proliferation (Huang et al., 2002). The differences in the
results between the study by Huang et al. (2002) and our
study might be due to the different materials and methods
(such as specimen preparation). In addition, materials
within the same category may not have behaved similarly. Variations in the release of ions occur in different
products (Sidhu and Schmalz, 2001). For instance, differences in the pattern and amount of fluoride released
were shown among different commercial products (Forss,
1993; Oliva et al., 1996; Kan et al., 1997).
Some studies have investigated the pulpal effects of
resin modified glass ionomers and reported different results (Sidhu and Schmalz, 2001; Gaintantzopoulou et al.,
1994). The different outcomes may be due to the disparities in the chemical compositions of the materials, particle
size, curing time and setting properties.
In vitro results extrapolating to the clinical situation
should only be done with extreme caution. Lack of simulation of the in vivo situation and the difficulties in extrapolating the data to the patient are the main limitations of
cell culture toxicity tests (Schmalz, 1994). Furthermore,
due to the lack of defense and repair mechanisms under
in vitro conditions, cells may show less tolerance to the
materials, which are biocompatible in vivo (Mller et al.,
1990).
Further studies are recommended to evaluate cytotoxicity of these restorative materials for longer period of
time to resemble clinical conditions. In addition, SEM
growth assay would seem to be useful in evaluating the
morphology of human gingival fibroblasts.
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