Basic ResearchTechnology

Preferential Attachment of Human Gingival Fibroblasts to

the Resin Ionomer Geristore
Fuwad Al-Sabek, DDS, MS, Sandra Shostad, DDS, MS, and Keith L. Kirkwood, DDS, PhD
Abstract
The resin ionomer Geristore has been used extensively
for root perforation repairs. The purpose of this study
was to evaluate oral in vitro biocompatibility of the
resin ionomer Geristore compared to two other dental
perforation repair materials, Ketac-Fil and Immediate
Restorative Material (IRM). Growth and morphology of
human gingival fibroblasts (HGFs) was determined using scanning electron microscopy (SEM) of HGFs cells
grown on test materials as well as cytotoxicity assays
using eluates from test materials. SEM analysis showed
that HGFs attached and spread well over Geristore with
relatively normal morphology. SEM showed that fibroblasts did not attach and spread well over Ketac-Fil or
IRM as cells appeared much fewer with rounded and
different morphology than fibroblasts grown on Geristore. Cytotoxicity assays indicated that HGFs proliferated in the presence of Geristore eluates and not in the
presence of Ketac-Fil or IRM eluates. In vitro interpretation indicates that Geristore is less cytotoxic to gingival fibroblasts.

From the Departments of Endodontics and Periodontics,

Oral Biology, and Pharmacology & Toxicology, State University
of New York at Buffalo, Buffalo, NY.
Address request for reprints to Keith L. Kirkwood, DDS,
PhD, Assistant Professor, Department of Periodontics/Prevention/Geriatrics, University of Michigan 1011 N. University Ave.,
Ann Arbor, MI 48109-1078; E-mail address: klkirk@umich.edu.
Copyright 2005 by the American Association of
Endodontists

adicular perforations can occur during Endodonontic treatment, postspace preparation, postremoval, and operative procedures. Perforations that occur below the
level of the epithelial attachment can cause infection, which may result in attachment
loss. These defects have been treated in the past with a variety of different materials
including gutta-percha, calcium hydroxide products, amalgam, Immediate Restorative
Material (IRM), cavit, and super Epoxy Benzoic Acid (EBA). Major problems relative to
these materials include biocompatibility and inadequate sealing. The use of composite
resins, glass ionomer cement and Mineral Trioxide Aggregate (MTA) has been suggested to circumvent these problems. Studies have shown that MTA is the most suitable
material for repairing sub-osseous perforations especially furcation perforations, because of its superior sealing and biocompatibility characteristics (1, 2), as well as
permitting mineralized matrix formation (3).
Subgingival root perforations that are supraosseous have been repaired using
glass ionomers and composite resins. However, all studies have demonstrated some
degree of cytotoxicity through in vitro and in vivo studies (4 14). Recently, resin
ionomers such as Geristore, originally designed for restorative procedures, have been
used in treating subgingival defects such as root resorption and perforation. Geristore
is a dual cure (both self and light-curing), hydrophilic, nonaqueous polyacid modified
composite resin composed of fluoride releasing glass, mainly barium fluorosilicate, and
a polymerizable organic matrix (Modified Bis-GMA, including 2-HEMA) combined with
a photoinitiator. Advantages of these materials include insolubility in oral fluids, increased adhesion to tooth structure, dual cure capabilities, low cure shrinkage, low
coefficient of thermal expansion, radiopacity, fluoride release, and biocompatibility
(15). Relatively few clinical studies have addressed biocompatibility (15), although
several clinical studies have demonstrated Geristore could repair subgingival and subosseous defects and could be used as a barrier for guided tissue regeneration (16 22).
Cytotoxicity of resin ionomers (Geristore) towards oral tissues has only started to be
investigated (23). The aim of the present study is to evaluate in vitro biocompatibility of
human gingival fibroblasts with Geristore using scanning electron microscopy and
nucleic acid fluorescent staining to measure cell attachment and morphology as well as
cell cytotoxicity assays to eluates from root perforation repair materials namely Geristore, Ketac-Fil, and IRM.

Materials and Methods

Cell Culture Conditions
Human gingival fibroblasts (HGF-1; American Type Culture Collection, Manassas,
VA; #ATCC CRL-2014) were obtained through commercial sources for these studies.
Cells utilized for these studies were between passages 14 and 21. Cells were cultured in
DMEM (Invitrogen Life Technologies, Grand Island, NY), supplemented with 10% fetal
bovine serum (Sigma, St. Louis, MO), penicillin (100 U/ml; Sigma), and streptomycin
(100 g/ml; Sigma) at 37C in a humidified atmosphere of 5% CO2 in air. The culture
medium was changed every 3 to 4 days.
Sample Preparation and Sterilization
Geristore (DEN-MAT Corporation, Santa Maria, CA.), Ketac Fil (ESPE, Seefeld/
Oberbay, Germany), and IRM (Caulk/Dentsply, Milford, DE) were obtained from commercial sources. Discs (6 2 mm) from the three materials were fabricated under
aseptic conditions by packing the material after mixing in a Teflon washer (internal
diameter of 6 2 mm), and compressed between two glass slides to generate even

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Figure 1. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on (A) glass coverslip controls, (B)
Geristore, (C) Ketac Fil, or (D) IRM. SEM views are shown at 100 with reference bar length indicated.

thickness of material. Glass cover slips were used as positive controls in

all experimental conditions throughout this study. Extracts from all
three materials were prepared by preincubating sterilized discs in 50
cm3 conical tubes with 5 ml of DMEM (Life Technologies) supplemented with 100 U/ml penicillin and 100 g/ml streptomycin at 37C in
a shaker (Labline, Barnstead Int., Dubuque, IA) at 150 rpm.

Scanning Electron Microscopy Growth Assay

Attachment, growth and morphology of human gingival fibroblasts
over Geristore were evaluated by scanning electron microscopy. Geristore,
Ketac Fil and IRM discs and control glass cover slips were sterilized as
described above and placed in the bottom of a 12-well culture plate. HGF-1
cells were seeded into the wells at density (3 104 cells per well) in DMEM
medium containing 10% fetal bovine serum. After incubation, cells were
washed three times with phosphate buffered saline and fixed in 2.5% glutaraldehyde in 1 mmol cacodylate buffer (pH 7.4). Samples were then
observed using a JEOL JSM 6400 scanning electron microscope (Jeol USA,
Peabody, MA). Cell attachment and viability in each experimental condition
were assessed by qualitatively comparing morphology of cells over tested
materials with that of cells over glass coverslips, which were considered
cells with normal morphology.
Cytotoxicity Assays
Human gingival fibroblasts cytotoxicity towards Geristore, Ketacfil, and IRM material extracts was determined by means of using the
CellTiter Aqueous Non-Radioactive Cell Proliferation Assay (Promega,
Madison, WI). HGF-1 cells were seeded into 96-well culture plates at
1 104 cells per well in DMEM supplemented with 10% fetal bovine
serum. After 24h cells were rinsed three times with PBS and treated with
100 l of the following material eluates in serum-free media: Geristore
(24 and 72 hr extracts), IRM (24 and 72 hr extracts), and Ketac-fil (24
and 72 hr extracts). Controls included media only (no material ex206

Al-Sabek et al.

tracts) and media without cells present. Soluble formazan absorbance

was recorded using an ELISA plate reader at 490 nm. These experiment
were repeated four times (n 4). Cytotoxicity was calculated by expressing absorbance values as a percentage of control values (100%
represented zero cytotoxicity). ANOVA statistical analysis was used to
assess if differences exist between controls and treatment groups and
the Tukey-Kramer test was used for multiple comparisons of means to
determine differences between treatment groups.

Results
Cellular Attachment and Growth Assays
For direct visual comparisons of gingival fibroblasts grown on
Geristore, Ketac-Fil, and IRM, we employed SEM analysis to view cell
morphology and spreading over 72 h in culture under serum-free conditions. As a control, HGF-1 cells attached and spread well over glass
coverslips in these studies (Fig. 1A). Compared to glass coverslips,
HGF-1 cells grew and spread qualitatively equally well over the surface
of Geristore exhibiting characteristic elongated fibroblastic morphology
(Fig. 1B). However, HGF-1 cells grown on coverslips appeared to have
a smoother cell membrane compared to cells on Geristore. However,
human gingival fibroblasts attached poorly to Ketac-Fil (Fig. 1C) and
IRM (Fig. 1D) under the same experimental concentrations. In addition, these experiments were repeated in the presence of serum (2 and
10%) to rule out any deleterious effects on HGF-1 cells because of the
absence of serum. As shown in Fig. 2, HGF-1 cells could attach to
Geristore in the presence of serum (Fig. 2A, B; 10 and 2% FCS, respectively) as well as in the absence of FCS (Fig. 2C). Cells grown on Geristore also had similar cellular morphology as glass coverslip controls
(Fig. 2D). In parallel experiments, HGF-1 cells failed to attach to either
Ketac-Fil or IRM in the presence of serum (data not shown).
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Basic ResearchTechnology

Figure 2. Scanning Electron Microscopic analysis of human gingival fibroblasts attachment and growth following 72-h incubation on Geristore under different serum
conditions. HGF-1 cells grown on Geristore in media containing (A) 10%, (B) 2%, or (C) 0% fetal calf serum (FCS) in tissue culture media. Panel D indicates HGF-1
cells grown on glass coverslip controls in 10% FCS containing media. SEM views are shown at 500 with reference bar length indicated.

Cytotoxicity Assay
To determine if Geristore was less cytotoxic to human gingival
fibroblasts, in vitro cytotoxicity (MTS) assays were performed using
material extracts with HGF-1 cells. Results obtained with the MTS assay
indicated that Geristore was less toxic to HGF-1 cells after 24 h incubation period than toxicity observed with Ketac Fil or IRM extracts (Fig. 3).
In these studies, we used serum free media as the control since 24 and
72 h material extracts were also prepared in serum free media (ascribed 100% viability). Normalized percent viability values from multiple experimental samples (n 4) were subjected to statistical analysis.
ANOVA statistical analysis indicated that material extracts had a significant effect on cell proliferation (cell cytotoxicity) with a p 0.0001
(F 13.105). Posthoc pair wise comparison using Tukey-Kramer multiple comparison test indicated that significant differences exist between
Geristore and both IRM and Ketac Fil. Results from Tukey-Kramer statistical analysis indicated that Geristore was significantly different than
0% FCS at 72 h (p 0.05) but not at 24 h. However, IRM effects on cell
viability was not significantly different from 0% FCS control at either 24
or 72 h time points (p 0.05). The effects of Ketac Fil extracts on
HGF-1 cell viability was not significantly different from controls at 24 h
(p 0.05) but was significantly different at 72 h (p 0.001). Significant differences between Geristore and the other materials existed as
well. See Fig. 3 to compare Geristore with Ketac Fil and IRM at 24 and
72 h time points. Significant differences between the same materials
using 24 and 72 h extracts were compared using Student t test. Geristore
(p 0.04) and Ketac Fil (p 0.0008) indicated significant differences
between material extracts but not for IRM (p 0.057).

Discussion
Materials used adjacent to oral tissues should have minimal cytotoxicity towards oral cells. Geristore is currently being used to repair

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subgingival defects and the interaction between Geristore in the defect

with adjacent periodontal tissues must be critical in the wound healing
process. Evaluation of cellular growth and attachment has been used to
test cytotoxicity of dental materials (4, 6, 10, 24, 25). It has been
suggested that cell growth on the surface of a material is a more sensitive
indicator of cytotoxicity than surrounding cell growth (5, 26). Scanning
electron microscopy has been used to evaluate adhesion of cells on
materials used in a proximity to periodontal tissues as a part of evaluating the cytotoxicity of these materials (27).
Geristore did not inhibit growth of gingival fibroblasts as evaluated
by scanning electron microscopy. Fibroblasts in this study grew and
spread well over Geristore with a morphology close to that of the controls. Interestingly, we found that fibroblasts attached well to Geristore
even in the absence of serum in the tissue culture media. When evaluated by the scanning electron microscopy, cells did not grow or spread
well over Ketac Fil or IRM. These cells appeared rounded and balled up
when compared with cells over Geristore and the controls in the presence and absence of serum (Figs. 1 and 2). Adhesion and spreading of
cells on a material surface are the initial phase for cellular function. The
persistence of rounded cells with little or no spreading suggests the
surface material may be toxic (28).
A recent study has also evaluated gingival fibroblast and periodontal ligament fibroblast attachment to Geristore (23). These investigators
determined that cellular attachment occurred significantly greater than
other endodontic root-end filling materials tested with approximately
90% attachment after 72 h. These studies are consistent with our
present study where we observed that gingival fibroblasts attached more
readily to Geristore than even glass cover slip controls. The same investigators also determined that gingival fibroblasts attached to Geristore in
an integrin-independent manner (23) indicating that integrins do not
directly mediate attachment to this material.

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References

Figure 3. HGF cell viability assay. Geristore, IRM, and Ketac Fil material extracts
were prepared following 24 (A) and 72 (B) hour incubation periods in serumfree tissue culture media. HGF-1 cells were incubated for 24 h in the presence
of material extracts and MTS cell proliferation assay was used to determine cell
viability in the presence of material extracts. Control cells were incubated in
serum free media in the absence of any material. Statistically significant differences (ANOVA, p 0.001) using posthoc Tukey multiple comparison are
indicated between control and test materials. Refer to text for details.

Regardless of the mechanism of cellular attachment to Geristore,

gingival fibroblasts appeared to have less cellular cytotoxicity in the
presence of extracts prepared from Geristore. We observed that gingival
fibroblasts proliferated significantly better (p 0.05) in the presence
of Geristore extracts compared to controls after 72 h in culture. Gersitore extracts were significantly less toxic to gingival fibroblasts than IRM
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It is unclear why Geristore has more favorable cellular response. It
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(23). Another reason could be that Geristore elutes less toxic materials
into the medium. Our current results support this conclusion, however,
the exact component differences are not known at the present time.
Several components of dental resin composite monomers or additives
are cytotoxic to fibroblasts, including gingival fibroblasts (10). Therefore, future studies will be conducted to evaluate the organic constituents present in Gersitore and compare it to other resin or perforation
repair materials and test their effects on different oral cells.

208

Al-Sabek et al.

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