Aust Endod J 2011; 37: 1825

ORIGINAL RESEARCH

Histological evaluation of rat tissue response to GMTA,

Retroplast, and Geristore retrograde lling materials
aej_195

18..25

Huda M. Hammad, BDS, MS, FRCD(C)1; Mohammed A. Hamadah, BDS, MS2*;

and Wael M. Al-Omari, BDS, MDentSci, PhD2
1 Department of Oral Medicine and Surgery, Faculty of Dentistry, Jordan University of Science and Technology, Irbid, Jordan
2 Department of Conservative Dentistry, Faculty of Dentistry, Jordan University of Science and Technology, Irbid, Jordan

Keywords
biocompatibility, Geristore, mineral trioxide
aggregate, retrograde lling, Retroplast.
Correspondence
Dr Huda M. Hammad, Department of Oral
Medicine and Surgery, Faculty of Dentistry,
Jordan University of Science and Technology,
P. O. Box 3030, Irbid 22110, Jordan. Email:
hmhammad@just.edu.jo
doi:10.1111/j.1747-4477.2009.00195.x
*Present address: Ayma Consultative Centre,
King Fahad Road, Opposite Kingdom Tower,
Riyadh, Kingdom of Saudi Arabia, Tel. 2795555.

Abstract
The aim of the study was to compare the short-term biocompatibility of grey
mineral trioxide aggregate (GMTA), Retroplast and Geristore. Silicon tubes
filled with the materials and empty control tubes were implanted in the dorsal
connective tissue of 30 Wistar albino rats. The tubes and surrounding tissues
were excised and prepared for histological examination at 1 week, 1 month
and 2 months after implantation. Inflammatory cell counts and the presence or
absence of necrosis adjacent to the materials and control tubes were recorded.
Data were statistically analysed using one-way ANOVA and Tukeys multiple
comparisons tests. The empty control tubes were well tolerated. All tested
materials showed a more severe initial reaction than the control group. With
time, the reaction became chronic, with variable increase in the numbers of
inflammatory cells. Retroplast recorded the most statistically significant
increase in the sum of inflammatory cells. Although the increase in the sum of
inflammatory cells was statistically significant for Geristore but not for GMTA,
the inflammatory cell counts for both were comparable. It was concluded that
the three materials continued to irritate tissues throughout the evaluation
period. Retroplast was the least biocompatible of the three tested materials at
2 months, followed by Geristore then GMTA.

Introduction
The need for biocompatible root-end filling materials
cannot be overemphasised. Currently available materials
have a variety of tissue responses. These materials are
advertised with claims of each possessing all the needed
features of an optimum retrograde filling material.
A wide range of root-end filling materials have been
investigated and used. These include amalgam, gutta
percha, zinc oxide-eugenol cements, mineral trioxide
aggregate (MTA), glass ionomer cement, composite
resins, compomers, Diaket and others (1,2).
MTA was developed at Loma Linda University for use
as a root-end filling material in surgical endodontic treatment. It is commercially available as ProRoot MTA (Tulsa
Dental Products, Tulsa, OK, USA), in a grey or white
form, or as MTA-Angelus (Angelus Solues Odontolgicas, Londrina, Brazil) (3). The MTA powder consists of
18

fine hydrophilic particles of tricalcium silicate, tricalcium

aluminate, tricalcium oxide, silicate oxide and other
mineral oxides which set upon hydration (4). It has been
used in pulp capping, root-end filling and perforation
repair (1). A considerable number of in vitro and in vivo
studies have assessed the biocompatibility of MTA (1,3).
Most of these studies indicate that MTA is a biocompatible
material, with favourable cellular or tissue responses
(1,3). A few studies have shown results not supportive of
its biocompatibility (3).
Retroplast (Retroplast Trading APS, Roervig, Denmark)
is a chemically cured two-component composite containing bisphenol-A-glycidyl-methacrylate (Bis-GMA)
and triethyleneglycol-dimethacrylate (TEGDMA) (2). It is
used for retrograde root filling in combination with a
dentine-bonding agent (5,6). Although many studies
have indicated the biotoxicity of its two main components, Bis-GMA and TEGDMA (711), a number of

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GMTA, Retroplast and Geristore

H. M. Hammad et al.

long-term clinical studies of Retroplast have shown high

success rates when applied under optimal conditions
(5,6).
The compomer or resin ionomer Geristore (Den-Mat
Corporation, Santa Maria, CA, USA) is a hydrophilic,
nonaqueous, polyacid-modified composite resin composed of fluoride releasing glass, mainly barium
fluorosilicate, and a polymerisable organic matrix (modified Bis-GMA, including 2-hydroxyethyl-methacrylate
(2-HEMA)) combined with a photoinitiator (12). An
easier to dispense version, Geristore Syringeable, is also
available. Reported advantages of resin ionomers 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 (1214). In addition to the
toxicity of Bis-GMA mentioned earlier, HEMA has also
been reported to be a biologically toxic material (7,11).
The cytotoxicity of polyacid modified composite resins
was found to vary considerably depending on the product
tested (15). However, some studies have indicated the
biocompatibility of Geristore (12,14,16) and clinical
success in repair of subgingival and subosseous defects,
and in guided tissue regeneration (13,14,1618).
The aim of the present study was to histopathologically
examine the biocompatibility of grey mineral trioxide
aggregate (GMTA), Retroplast and Geristore by implanting them into subcutaneous connective tissue of rats.

Materials and methods

Thirty male Wistar Albino rats, 34 months old, weighing
300350 g were used. The animal care was provided by
the animal house facility at the Biomedical Research
Center, Jordan University of Science and Technology. All
surgical procedures were performed at the same facility.
The study protocol was approved by the Jordan University of Science and Technology Deanship of Research
and the University Animal Care and Use Committee. The
rats were anaesthetised using a regimen consisting of
atropine (0.020.05 mg kg-1, ketamine hydrocloride (40
87 mg kg-1) and xylazine hydrochloride (513 mg kg-1).
Animals were later euthanised using an anaesthetic overdose. All procedures and animal care were carried out in
accordance with the guidelines laid down by the National
Institute of Health, USA (19), and the Canadian Council
on Animal Care (20).
Silicon tubes (Deutsch & Neumann, GmbH, Berlin,
Germany) with a 2-mm inner diameter and 4-mm outer
diameter, were cut into 7-mm pieces and autoclaved. For
each animal, three tubes were used as carriers for the
three tested materials after being prepared according to

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Australian Endodontic Journal 2009 Australian Society of Endodontology

the manufacturers instructions. The following materials

were investigated:
GMTA (Dentsply Tulsa Dental, Tulsa, Johnson City,
TN, USA).
Retroplast (Retroplast Trading APS, Roervig,
Denmark).
Geristore Syringeable (Den-Mat Corporation, Santa
Maria, CA, USA).
An empty tube served as the control. The tubes were
implanted dorsally in the subcutaneous connective
tissues of the rats. The dorsal skins of animals were
shaved and disinfected using 5% iodine solution, 2-cm
horizontal incisions at least 2-cm apart were made on
each side of the dorsal midline of each rat, using a no. 15
blade (Aesculap AG and Co. KG, Tutlingen, Germany). A
pocket-like space was created in the skin by blunt dissection into which silicon tubes were implanted. The skin
was closed with 3/0 silk suture.
Ten animals were sacrificed at three examination
periods: 1 week, 1 month and 2 months. After shaving
the areas containing the implants, the connective tissues
containing the implants were excised. The specimens
were kept in 10% formalin for 1 week. Sections of 5 mm
thickness were taken from specimens embedded in paraffin blocks and stained with hematoxylin and eosin
for histological evaluation. These sections were studied
qualitatively and quantitatively using a light microscope
(Olympus, U-MDOB BX 40, Olympus optical Co. LTD,
Japan) at 40, 100 and 400 magnifications. The
observer was blinded to the group origin of the tissue.
An average value for each group was obtained from the
sum of all inflammatory cells counted in 10 randomly
selected separate areas (21). Fibroblasts, blood vessels
and inflammatory cells (polymorphonuclear leukocytes,
plasma cells, lymphocytes, macrophages and giant cells)
were also counted.
The inflammatory reactions were also rated and scored
as: 0, none or few inflammatory cells (no reaction); 1, <25
cells (mild reaction); 2, between 25 and 125 cells (moderate reaction); 3, 125 cells (severe reaction) (22). The
presence or absence of suppuration or necrosis was
also noted.
Results were statistically analysed with one-way ANOVA
test and Tukeys multiple comparisons test for comparison
of severity of inflammation at intervals and for materials.

Results
Control group
Gradually decreasing mild inflammation was observed at
the tube ends throughout the three test periods, with
ingrowths of granulation tissue detected inside the tubes
(Fig. 1). Pronounced fibroblastic activity and angiogenesis
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GMTA, Retroplast and Geristore

H. M. Hammad et al.

test periods. Liquefactive necrosis could be detected in

seven cases at the 1-week period (Fig. 4), including one
severe case, and in one case each at the 1-month and
2-month periods. A well-developed fibrous capsule could
be seen around the tubes at the 1-month and 2-month
periods. Chronic granulomatous inflammatory reaction
was seen in seven cases at the 2-month period with
apoptotic giant cells containing material particles (Fig. 5).
Small ingrowths of granulation tissue were seen in the
tube ends adjacent to the material.
A statistically significant increase was observed in the
sum of the inflammatory cells among the three test
periods (P < 0.05, Table 1).

Geristore group

Figure 1 Mild inammatory response with an ingrowth of granulation

tissue (I) inside the tube (T) at the 1-month period in the control group. A
well-developed brous capsule (C) is seen (original magnication 50).

were noted at the 1-week period. A thin, well-developed

fibrous capsule could be seen around the tubes at the
1-month period (Fig. 1), becoming dense at the 2-month
period. No necrosis was observed at the three test periods.
The mean value of the sum of the inflammatory cells
for the control group at all three periods showed no
significant difference (P > 0.05, Table 1).

GMTA group
Gradually increasing chronic inflammation was observed
at the tube ends throughout the three test periods,
ranging between mild at the 1-week period, and mild to
moderate at the two other periods (Fig. 2). Mild liquefactive necrosis was noted adjacent to GMTA in four cases at
the 1-week period only (Fig. 3). It was not uncommon to
see GMTA particles extruded into tissue, evoking a
foreign body reaction at all test periods (Fig. 2). Active
angiogenesis was clearly noted adjacent to the tube ends.
A fibrous capsule could be seen around the tubes at
the 1-month period, becoming well-developed at the
2-month period.
Although there was an increase in the mean values of
the sum of the inflammatory cells throughout the three
test periods, the increase was not statistically significant
(P > 0.05, Table 1).

Retroplast group
Gradually increasing mild to moderate chronic inflammation was observed at the tube ends throughout the three
20

Gradually increasing chronic inflammation was observed

at the tube ends throughout the three test periods,
ranging between mild at the 1-week period, and mild to
moderate at the two other periods with birefringent
material particles extruded into the tissue seen in some
cases (Fig. 6). Focal liquefactive necrosis was observed in
the areas immediately adjacent to the tube ends in six
cases at the 1-week period (Fig. 7) and in only one case at
the 1-month period. A well-developed fibrous capsule
could be seen around the tubes at the 1-month period,
becoming dense at the 2-month period.
There was a statistically significant increase in the
mean value of the sum of inflammatory cells for Geristore
at the three test periods (P < 0.05, Table 1).

Comparison among the four groups at the

1-week period
The one-way ANOVA test showed a statistically significant
difference in the sum of the inflammatory cells among the
groups (P < 0.05, Table 1). Tukeys multiple comparisons
test used to determine the differences among the four
groups revealed that the only statistically significant
difference was between the GMTA and control groups
(P < 0.05). The latter recorded the lowest mean value of
the sum of inflammatory cells, followed by the Geristore
group, the Retroplast group, then the GMTA group
(Table 1).

Comparison among the four groups at the

1-month period
The one-way ANOVA test showed a statistically significant
difference in the sum of the inflammatory cells among the
groups (P < 0.05, Table 1). Tukeys multiple comparisons
test revealed a significant difference between the control
group and each of the other groups, the difference being

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GMTA, Retroplast and Geristore

H. M. Hammad et al.

Table 1 Mean value and standard deviation of the sum of inammatory cells for all materials at all test periods
mean SD (n = 1)
Material

1 week

1 month

Control
Retroplast
Geristore
GMTA
p Value

12.000 4.807
17.200 5.095
14.500 3.719
19.400 3.534a
0.003
a

2 months

10.300 5.057
23.800 9.343b
22.600 10.047c
22.700 8.260d
0.002

bcd

P value

8.400 2.951
35.300 18.670e
24.300 10.220f
25.500 8.317g
0.000
efg

0.202
0.010
0.036
0.174

Signicant P values with ANOVA test are shown in bold font. Values carrying the same letter superscript within the same period showed signicant
differences (P values) with Tukeys multiple comparisons test.
GMTA, grey mineral trioxide aggregate.

Figure 2 Moderate chronic granulomatous inammation at the tube

ends adjacent to GMTA at the 2-month period. It was not uncommon to
see GMTA particles extruded into the tissue seen here as a dark material
(original magnication 100).

most significant from Retroplast, followed by GMTA, then

Geristore (P < 0.05, Table 1).

Comparison among the four groups at the

2-month period
The one-way ANOVA test showed a statistically significant
difference in the sum of the inflammatory cells among the
four groups (P < 0.05, Table 1). Tukeys multiple comparisons test revealed a significant difference between the
control group and each of the other groups, the difference
being most significant from Retroplast, followed by
GMTA, then Geristore (P < 0.05, Table 1).
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Australian Endodontic Journal 2009 Australian Society of Endodontology

Figure 3 Liquefactive necrosis adjacent to GMTA at the 1-week period

(original magnication 200).

Discussion
It is generally accepted that using small animals in
implantation tests is suitable to provide preliminary data
on the local effects of the implanted materials (21).
Various tube materials have been used to hold the
tested materials such as dentine tubes (23), Teflon tubes
(24), polyethylene tubes (25) and silicon tubes (21). Use
of dentine tubes carries the risk of introducing immunologically foreign proteins into animal tissues and the need
to be instrumented to a standard size (24). We have used
silicon tubes to avoid some methodological problems and
foreign body reactions that have been reported with polyethylene and Teflon tubes but not with silicon tubes (21).
As for the inflammatory tissue response at the 1-week
period, it is expected to be partly caused by the surgical
21

GMTA, Retroplast and Geristore

Figure 4 Moderate chronic inammation adjacent to Retroplast at the

1-week period with necrosis (N), (original magnication 50).

H. M. Hammad et al.

Figure 6 Moderate chronic inammation adjacent to Geristore at the

2-month period, with birefringent material particles (B) extruded into the
tissue (original magnication 400).

Figure 5 Chronic granulomatous inammatory reaction adjacent to Retroplast at the 2-month period with apoptotic giant cells (A) containing
material particles (original magnication 200).

Figure 7 Focal liquefactive necrosis (N) adjacent to Geristore at the

1-week period (original magnication 50).

trauma produced during the placement of the tubes (21),

in addition to the toxic effects of the implanted materials,
including tube material. As wound healing takes place, it
is logical to think that the reaction remaining at the 1and 2-month periods is to the implant and tube materials.

Although the decrease in the mean value of the sum of

inflammatory cells in the empty silicon tubes or control
group was not statistically significant throughout the
three test periods, the inflammation was initially mild,
and all types of inflammatory cells showed a decrease in

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H. M. Hammad et al.

their counts between the 1-week and 2-month periods.

No foreign body reaction was noted with the empty
silicon tubes, and with time, a well-organised fibrous
capsule was present. These findings are similar to those of
previous studies (21), and support the choice of silicon
tubes as carriers for testing the biocompatibility of different materials.
The case was not the same for the three tested materials, which all showed an increase in inflammatory cell
counts with time. When the material groups were compared at various test periods, the differences in mean
values of inflammatory cell counts were not significant,
but when they were compared with the empty control
group, all materials showed significantly higher counts at
the 1-month and 2-month periods. Only GMTA showed
counts significantly higher than the control at the 1-week
period.
GMTA initially evoked moderate to severe inflammation, accompanied by foreign body reaction and liquefactive necrosis. This picture tended to resolve with time in
half of the cases; as necrosis was totally absent at the
2-month period. The same findings were previously
reported (23). Moretton et al. also reported similar findings and stated that the initially severe inflammatory
response to GMTA is most likely multifactorial (26). The
factors include high pH and heat generated during the
setting reaction; because the calcium oxide component,
when mixed with water, generates calcium hydroxide in
an exothermic reaction. Generation of inflammatory
cytokines such as interleukin-1 and interleukin-6 contributes to the process (26). De Morais et al. found persistent moderate chronic inflammation around MTA at
60 days post implantation (27). They attributed this persistence to the extrusion of MTA out of the tube, which
favoured the accumulation of macrophages in the area.
Yaltirik et al. also recorded comparable findings at
60 days, while at 90 days, there was no inflammatory
infiltration (28). The current study agrees with these
findings, and this may be attributed to the consistency of
MTA, which demonstrated detachment of small particles
from the main material mass into the surrounding tissue,
evoking a foreign body reaction. Therefore, in clinical
practice, care must be taken to leave the surgical apical
field as clean as possible after using MTA as a root-end
filing material.
Retroplast caused considerable necrosis in some cases
at all three test periods, in comparison with Geristore,
which caused more limited necrosis in a few cases at the
1-week and 1-month periods, and GMTA, which caused
mild necrosis only at the 1-week period. The chronic
inflammatory reaction was most pronounced with Retroplast at the final assessment, with granulomatous reaction seen in the immediate vicinity of the material.

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Australian Endodontic Journal 2009 Australian Society of Endodontology

GMTA, Retroplast and Geristore

Furthermore, Retroplast showed the highest mean value

of the sum of inflammatory cells, followed by GMTA,
then Geristore. The ingrowth of granulation tissue in the
tube ends indicates shrinkage of the material.
Retroplast is primarily composed of Bis-GMA and
TEGDMA (2). Synergistic cytotoxic effects have been
observed when combinations of TEGDMA and Bis-GMA
were tested (7), but a significantly lower concentration of
Bis-GMA was needed to suppress cellular growth than
the co-monomer TEGDMA (10). However, only trace
amounts of the hydrophobic, bulky Bis-GMA were found
to leach into aqueous media, compared with the relatively small, hydrophilic co-monomer TEGDMA which
was found to leach in larger quantities (8). Thus, leaching
of TEGDMA into the nearby humid connective tissue
environment may explain the higher incidence of necrosis with Retroplast, especially at the initial assessment
period, with the effects becoming milder as the toxic
material is neutralised and removed by local lymphatic
drainage (29).
Rud et al. reported high success rates with Retroplast
in long-term clinical studies (5,6). An important step in
their retrograde filling technique consists of washing the
surface, 3 minutes after curing of the Retroplast, with
96% ethanol, in an attempt to remove the unpolymerised
surface layer (5). This is important to reduce the amount
of toxic leachable components and thus reduce the cytotoxicity. In our study, an ethanol wash was not used. This
may have contributed to the different results indicative of
lower biocompatibility.
Geristore contains Bis-GMA and HEMA (12), both of
which have been found to be cytotoxic (7). The hydrophilic monomer HEMA was found to be the chief
constituent released from compomers (15). The cytotoxicity of HEMA was found to be much less than
that of TEGDMA, which in turn was less cytotoxic
than Bis-GMA (7). This explains the relatively milder
reaction to Geristore compared with Retroplast in this
study.
The findings in this study regarding Geristore are not in
disagreement with those of Souza et al. who evaluated the
cytotoxicity of three contemporary resin modified glass
ionomer cements (RMGICs) or compomers, both in vitro
by cell culture, and in vivo by subcutaneous implantation
in rats (29). Despite variation of cytotoxicity in vitro, all
three materials significantly decreased cell metabolism
compared with the control group. In the implantation
experiment, complete healing at 90 days occurred only in
half of the samples of each material, and the remaining
samples showed mild to moderate inflammatory reaction.
They attributed the initial reaction to the slow acid-base
reaction that occurs in RMGICs that maintains a low pH,
and the interference of oedema with the monomer23

GMTA, Retroplast and Geristore

polymer conversion, with displacement of uncured resin

components such as HEMA to a great distance from the
implants. They speculated that low molecular weight
components such as HEMA might be removed by lymphatic drainage, and that the local tissue oedema neutralises the low pH, which results in decrease of
inflammation intensity that occurred with time. In cases
where complete resolution did not occur, large components (such as Bis-GMA) that are not removed by lymphatic drainage or digested by macrophages remained
in connective tissue, triggering the persistent chronic
inflammatory reaction. The same explanation may be
used in this study for the persistence of chronic inflammation, because there was a significant increase in macrophages and giant cells.
Ozbas et al. studied the reaction of rat connective tissue
to two compomers (Dyract and F2000), a composite, and
amalgam using polyethylene tube implants at 7, 15, 30,
60 and 90 days (30). Histological evaluation showed that
all materials created moderate or severe inflammatory
responses initially, which decreased by the 60th and
90th day. However, there were still significantly higher
numbers of inflammatory cells in experimental groups
compared with the control group. In this study, there was
a significant increase in the mean values of the sum of the
inflammatory cells at the 1-week and 2-month periods in
the Geristore group. This difference from Ozbas study
may be attributed to difference in the compomer used,
and the shorter time of the experiment.
Because later harmful effects of a material are considered to be more important than its initial effects (28), the
results of the histometric evaluation indicate that the
least favourable tissue reaction was observed with Retroplast, followed by Geristore then GMTA. Although the
mean value of the sum of inflammatory cells after
2 months was almost similar for GMTA and Geristore, the
increase in the sum of inflammatory cells was statistically
significant for Geristore but not for GMTA. Furthermore,
Geristore showed a higher standard deviation than
GMTA.

Conclusions
Within the limitations of the current study, the findings
indicated that GMTA, Retroplast and Geristore retrofilling
materials continued to irritate tissues, to variable degrees,
throughout the 2-month testing period. All tested materials caused a persistent foreign body reaction with the
macrophages and/or giant cells observed engulfing particles released from the implanted materials.
Retroplast was found to be the least biocompatible of
the three materials at 2 months, followed by Geristore
then GMTA.
24

H. M. Hammad et al.

Acknowledgements
This study was supported by grant no. 102/2007, Jordan
University of Science and Technology, Irbid, Jordan.

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