Evaluation of the Antimicrobial and Cytotoxic Activity of Epiphany ...

Egyptian Journal of Medical Microbiology, January 2007 Vol. 16, No. 1 

Evaluation of the Antimicrobial and Cytotoxic Activity of Epiphany 

Root Canal Sealer in Vitro 

Amany E. Badr 1 , Amro A Abdul Razek 2 , Nesrene S. Omar 3 , and Farid A.Badria 4 

1 Lecturer of Endodontics, Conservative Dentistry Department, 2 Assistant Professor of Endodontics, 

Conservative Dentistry Department, 3 Associate Professor of Medical Microbiology and Immunology, 

Faculty of Medicine, Mansoura University, 4 Professor of Pharmacognosy , Faculty of Pharmacy, 

Mansoura University 

This study evaluated the antimicrobial and cytotoxic effect of Epiphany root canal sealer 

versus commonly used sealers; EndoFill, Apexit, Ketac-Endo, and AH-26. The first portion of this 

study used four different microorganisms; Enterococcus faecalis (E.faecalis), staphylococcus aureus 

(S. aureus), Ecshericia coli (E.coli) and Candida albican (C. albican) to determine the antimicrobial 

effect using the agar-well diffusion method. Secondly, In vitro Cytotoxicity assay using human 

periodontal ligament cell culture was used to evaluate the cytotoxicity of Epiphany sealer compared 

to the other four sealers. The cell cultures were incubated for 24h with freshly mixed material and 

after 24 h of setting. The viability of the fibroblast cells was determined using trypan blue. The 

results of this comparative study indicated that Epiphany sealer exhibited a potential antibacterial 

activity with no anticandida effect. The cytotoxicity of Epiphany was comparable to that of 

EndoFill and Apexit, however it was significantly less toxic than Ketac-Endo, and AH-26. 

INTRODUCTION 

The role of bacteria and their byproducts 

in the initiation and perpetuation of 

pulp and periapical diseases has been well 

established. Most infecting bacteria, together 

with their principal substrate of necrotic pulp 

debris, may be removed by routine 

endodontic procedures such as 

instrumentation, irrigation, and the use of an 

intracanal medicament with antimicrobial 

activity 1 . However, infection may persist in 

canals with high anatomic complexity, with a 

great number of bacteria, especially 

facultative anaerobic bacteria 2 . In addition, 

several studies have associated the presence 

of fungi with therapy-resistant endodontic 

infections 3 . 

After the microbial control phase of 

endodontic therapy, a root canal filling is 

placed to seal the root canal system from the 

external environment 3. The standard method 

of obturation of the root canal space is by 

using a core material in combination with a 

root canal sealer 4 . Antimicrobial activity 

plays an important role in the efficacy of an 

endodontic sealer during root canal filling, 

and for this reason many studies have dealt 

with the antibacterial activity of endodontic 

sealers 5 . 

Facultative microorganisms such as 

E. faecalis, E.coli and S.aureus and even 

C.albicans have been considered to be the 

most resistant species in the oral cavity and 

possible cause of failure of root canal 

treatment 6 . The agar diffusion method has 

been widely used to test the antimicrobial 

activity of dental materials and medications, 

the advantage of this method is that it allows 

direct comparisons of root canal sealers 

against the test microorganisms, indicating 

which sealer has the potential to eliminate 

bacteria in the local microenvironment of the 

root canal system 7 . 

Good tissue compatibility is 

decisive for root canal sealers because they 

may come into direct contact with tissues 

specially when extruded to the apical area 8 . 

Toxicity testing of dental materials can be 

assessed either in vitro or in vivo. Using cell 

lines is a common method of testing dental 

materials that allows for a simple, 

reproducible result that can be controlled in a 

laboratory setting. In vitro testing also allows 

for the comparison between several materials 

using the same cells under the same 

conditions 9 . 

Improvements in adhesive 

technology have fostered attempts to reduce 

apical and coronal leakage by bonding to 

canal walls 10 . These improvements rely on 

the incorporation of resin monomers into the 

sealer or application of resins during a distinct 

conditioning step. Other strategies have 

focused on substitutes for gutta percha that 

bond to the root dentin, thereby establishing a 

so called monoblock-obturation 11 . 

95


The Epiphany obturating system 

(Pentron Clinical Technologies, Wallingford, 

CT) which is a dual curable dental resin 

composite sealer uses Resilon points and is 

bonded to the root dentin 12 . Resilon (Resilon 

Research LLC, Madison, CT), a thermoplastic 

synthetic polymer based root canal filling 

material, has been developed that performs 

like gutta-percha, has the same handling 

properties, and for retreatment purposes may 

be softened with heat or dissolved with 

solvents like chloroform. Based on polymers 

of polyester, Resilon contains bioactive glass, 

bismuth oxychloride and barium sulfate 12 . 

Resilon associated with Epiphany has 

recently been introduced. Some of its 

mechanical and chemical properties have 

been evaluated with controversial results 11, 13 . 

In contrast, the biological properties of 

Resilon and Epiphany are not well 

documented 14 . 

The properties of root canal 

cements can be divided into physicochemical, 

antimicrobial and biological 15 . Therefore, the 

aim of this study was to in vitro evaluate the 

antimicrobial and cytotoxic properties of 

Epiphany sealer in comparison with the 

commonly used root canal sealers. 

MATERIALS AND METHODS 

The various root canal sealers that 

were tested in the present study are the 

adhesive resin root canal sealer (Epiphany)†, 

ZOE based sealer (EndoFill)‡, calcium 

hydroxide based sealer (Apexit)*, glass 

ionomer based sealer (Ketac-Endo)§ and 

Resin sealer (AH-26) ‡. 

†Pentron clinical technologies 

‡DentSply 

*Ivoclar Vivadent 

§3M ESPE 

Antimicrobial test 

Agar-well diffusion method (AWDM): 

Cultures of E. faecalis , S. aureus, 

E.coli and C. albican were obtained from 

endodontic isolates. All microorganisms 

were previously subcultured in appropriate 

culture plates and under gaseous conditions 

to confirm their purity. From the broth 

culture suspensions of the tested 

microorganism 0.2 ml were prepared 

adjusted to No. 0.5 McFarland scale were 

spread on four Petri dishes containing 

Mueller-Hinton Agar medium. Five wells of 

5mm depth and 4mm diameter were 

punched in the agar plates and filled with the 

test sealers (mixed according to 

manufacturer instructions). The plates were 

incubated aerobically at 37Cº for 48h. The 

diameters of the zones of microbial 

inhibition around each well were measured 

in millimeters after the incubation period. 

The inhibitory zone was considered to be the 

shortest diameter from the outer margin of 

the well to the initial point of the microbial 

growth. 

Three replicates were measured for 

each microorganism and a mean diameter was 

determined for each sealer. Greater diameters 

of zones of inhibition were interpreted to 

indicate greater antimicrobial activity of the 

involved sealers 16, 17 . 

Cytotoxicity assay 

Test materials and specimen preparation: 

The tested materials were mixed 

according to the manufacturer's instructions. 

Freshly mixed materials were filled in 

polyethylene rings of 5mm inside diameter 

and 4mm in height. The test was performed 

on fresh mix and 24h set mix. To prevent 

contamination of the set samples they were 

exposed to UV light for 2h before performing 

the test. In a pilot study made previously 18 , 

this period was sufficient to sterilize 

experimental materials. 

Cell Culture: 

Periodontal ligament cells (PDL) 

were obtained from teeth extracted for 

orthodontic purposes. Fibroblasts were grown 

in Dulbecco's modified Eagles medium 

(DMEM) supplemented with 10% fetal 

bovine serum and antibiotics (10,000 units of 

penicillin-G/ml, 10 mg of streptomycin/ml 

and 20 mM of L-glutamine) 19 . 

Growth Measurement: 

The fibroblast cells were plated at a 

density of 3-4 x10 4 cells/ml and dispensed 

onto 96-well culture plate with 1ml of 

medium per well and incubated at 37Cº 

supplemented with 5% CO 2 for 48h to allow 

96


attachment of the fibroblasts to the bottom of 

the wells 20 . Specimens of different sealers 

were placed onto the wells of the culture plate 

and each specimen was covered by 100µl 

suspension of fibroblasts and incubated at 

37Cº with 5% CO 2 for 48h. At the end of the 

incubation period, the culture medium was 

aspirated and 100µl of formaldehyde 

phosphate buffered saline (FPBS) was added 

to each culture well for 2h. FPBS was then 

removed and each well was thoroughly rinsed 

with distilled water and 100µl of 0.25% of 

trypan blue (wt/vol) was added to each well to 

stain the nonviable cells. After 3h, the trypan 

blue was removed and the cells were 

thoroughly rinsed with distilled water. The 

number of viable cells was expressed by 

averaging of six readings using an inverted 

microscope (Olympus, 1X71, Japan) 19 . 

Statistical analysis was performed by one way 

ANOVA, and Bonferroni tests. 

RESULTS 

Table 1 shows the mean of the 

zones of inhibition of microbial growth of 

each sealer against the microorganisms tested 

measured in mm and the average values of the 

antimicrobial activity of each sealer against 

all microorganisms. EndoFill produced the 

largest inhibitory zone followed by Epiphany 

(by average values). On the other hand, 

Apexit produced the smallest inhibitory zones 

against the tested microorganisms (by average 

values). It appears that Candida albican was 

the most resistant organism to the effect of the 

sealers in this experiment. Epiphany had the 

largest inhibitory zone on E.faecalis followed 

by S. aureus and E.coli. However, Epiphany 

had no effect on C. albican. Figures 1-4 show 

the zones of inhibition of the tested sealers 

with the different microorganisms. 

Table 1: Mean values of antimicrobial activity of root canal sealers against microorganisms 

tested (mm): 

E. faecalis 

S. aureus 

E. coli 

C. albicans 

Average inhibition zone of 

sealers for all 

microorganisms 

Epiphany 

9 

6 

5 

0 

5 

EndoFill 

7 

9 

6 

7 

7.25 

Apexit 

1.3 

5 

0.66 

0 

1.74 

Ketac‐ 

Endo 

0 

7 

4 

0 

2.75 

AH‐26 

6 

5 

4 

0 

3.75 

97


1 2 

2 

1 

3 

3 

5 

4 

5 

4 

tested sealers with E.faecalis 

Fig. 1: Inhibition zones of the 

Fig. 2: Inhibition zones of the tested 

sealers with S. aureus 

1 

2 

1 

2 

5 

3 

5 

3 

4 

4 

Fig. 3: Inhibition zones of the 

tested sealers with E.coli. 

١ 

1)Epiphany 2)EndoFill 3)Apexit 4)Ketac-Endo 

٢ 

5)AH-26 

Fig. 4: Inhibition zones of the tested sealers 

with C. albican. 

٢ 

١ 

٣ 

٥ 

٥ 

٤ 

٣ 

98


Table 2: Mean and standard deviation of viable cells after exposure to different sealers at 0-h (fresh) and 24-h. 

0-h 24-h 

Material mean value SD mean value SD 

Epiphany 28.1 a 0.4 30.5 a 0.83 

EndoFill 27.3 a 1.36 29.3 a 1.36 

Apexit 29.8 a 2.71 32.6 a 1.63 

Ketac-Endo 18.8 c 0.752 22.3 c 3.26 

AH-26 19.3 c 1.21 25.6 c 0 .516 

--------------------------------------------------------------------------------------------------------------- 

Values are means ± SD. Different superscript letters (a-c) indicate groups that are significantly 

different (p≤0.05). Groups identified with the same superscript letters are not significantly 

different (p>0.05). 

35 

30 

25 

% of viability. 

20 

15 

10 

0 h 

24 h 

5 

0 

Epiphany EndoFill Apexit Ketac- 

Endo 

Root canal Sealers 

AH-26 

Fig. 5: Histogram representing the Percentage of fibroblast cells viability after exposure to 

different sealers 

99


The results of cytotoxic effect 

evaluation of the examined sealers on 

fibroblast cells are shown in Table 2 and 

Figure 5. Although every material tested 

showed some degree of toxicity, the 

percentage of cell viability was greater with 

Epiphany, EndoFill and Apexit than with 

other sealers with no statistically significant 

difference between these three sealers in both 

time periods (fresh and 24 h). Other materials 

(Ketac-Endo, AH-26) showed significantly 

lower percentage of cell viability. All sealers 

showed increased number of viable cells after 

24 h. 

DISCUSSION 

A number of approaches have been 

used to evaluate the antimicrobial 

effectiveness in the laboratory. These include; 

incubation of broth cultures of the selected 

microorganisms with the agent under the test, 

growth of the selected microorganisms as 

lawns on agar surfaces using the disc 

diffusion method, and the artificial infection 

of extracted teeth with the selected organisms 

and irrigation with the test agent 21 . 

The agar diffusion method used in 

this study is one of the most often used 

methods for the antimicrobial activity 

assessment. The results of this method do not 

depend only on the toxicity of the material for 

a particular microorganism, but also on the 

diffusibility of the material across the 

medium. However, great care has been taken 

in this study to keep the plates for 2h at room 

temperature to allow the diffusion of the 

agent through the agar and then incubated at 

37ºC under appropriate gaseous conditions. 

The present study tested the antimicrobial 

activity of different sealers against 

microorganisms considered to be resistant to 

endodontic treatment. Therefore, if a sealer is 

effective against these microorganisms, it will 

probably be effective against the most 

susceptible ones 22 . 

In this study, Epiphany produced a 

clear antibacterial effect against the tested 

bacteria especially the most resistant one 

(E.faecalis). On the contrary, Bodrumlu and 

Semiz 23 showed that Epiphany root canal 

sealers had little effect on E.faecalis 23 . This 

can be explained by a research conducted by 

Gomes et al. 5 , who determined that the 

antimicrobial activity depends on the 

sensitivity of the drug, bacterial source (wild 

strains or collection species), number of 

bacteria inoculated, pH of the substrates in 

plates or tubes, agar viscosity, storage 

conditions of the agar plates, incubation time 

and the metabolic activity of the 

microorganisms. 

The results of this study showed 

that Epiphany had no effect on C.albicans. 

Moderate inhibition was produced by 

Epiphany on E.coli and S. aureus isolates. 

EndoFill had the maximum average 

zones of inhibition as compared to other 

tested sealers. However, it comes next to 

Epiphany regarding the effect against 

E.faecalis. On the other hand, this material 

was the only effective sealer on C.albican. 

Previous studies indicated that root canal 

sealers containing ZOE have a strong 

antibacterial effect 24 . This effect was due to 

the action of eugenol 25 . Kaplan and others 

have stated that the most effective 

antimicrobial sealers contain eugenol and 

formaldehyde 26 . 

Other endodontic sealers tested 

(Apexit, AH-26 and Ketac-Endo) were less 

effective than Epiphany in killing the 

microorganisms. The antimicrobial activity of 

Apexit may be based on its content of calcium 

hydroxide. Root canal sealers integrated with 

calcium hydroxide have enhanced 

antimicrobial activity 23, 27. The 

antimicrobial effect of this sealer is produced 

by the release of hydroxyl ions which 

increases the pH above 12.5 23. In this 

experiment Apexit had the lowest average 

diameter of inhibition zones. Bystrom and 

Sundqvist 28 also found that for a calcium 

hydroxide based sealer to be an effective 

antimicrobial agent, it should maintain a pH 

level greater than 12.5. As the calcium 

hydroxide sealer sets, the pH declines rapidly 

to 9.4 causing loss of effectiveness. 

Some endodontic sealers consists of 

polymer materials such as AH-26 which 

release small amount of formaldehyde during 

the polymerization process, and it is this agent 

that gives the resin based sealer its 

antimicrobial effect 29. Bodrumlu and Semiz 

23 showed that AH-26 had the lowest 

antibacterial effect against E.faecalis, and 

attributed this effect to the release of a small 

amount of formaldehyde over a brief period 

of time. In this study AH-26 had a minor 

effect against the tested organisms and in 

100


particular, it had a moderate effect on 

E.faecalis. 

The antimicrobial activity of Ketac- 

Endo is assumed to rely on fluoride ion 

release, but the quantities of fluoride released 

may not be sufficient to reach a concentration 

that effectively kills microorganisms 30. A 

weak antimicrobial activity of Ketac-Endo 

has been reported by Heling and Chandler 31. 

Antimicrobial activity of root 

canal sealers helps destroy the remaining 

bacteria. On the other hand, severe toxicity of 

a filling material may be a reason for damage 

of the periapical tissue thereby abolishing the 

beneficial effects of the antimicrobial 

properties of the material 

22 . The 

antimicrobial components of the sealer do not 

have selective toxicity against 

microorganisms; they also exert toxic effects 

on host cells 31 . 

Azar, et al 32 thought that in general, 

the toxicity of newly developed materials 

should be assessed using the three steps 

approach. A first step is to screen a candidate 

material using a series of in vitro cytotoxicity 

assays using cell cultures. Then, if the 

material is determined not to be cytotoxic in 

vitro, it can be implanted in subcutaneous 

tissue or muscle and the local tissue reaction 

evaluated. Finally, the in vivo reaction of the 

target tissue versus the test material must be 

evaluated in human subjects or animals. 

Therefore, if the test material induces a strong 

cytotoxic reaction in cell culture tests, it is 

very likely also to exert toxicity in living 

tissue. A reduction in the number of animal 

tests and the resulting expenses might be an 

additional benefit of such a screening 

approach. 

In this study, the cytotoxicity was 

evaluated by measuring the cell growth via 

cell counting. This method was chosen 

because it is easy, not requiring complicated 

or expensive equipments. 

In this present study, an in vitro 

cytotoxicity tests was done by using primary 

cells, mainly oral fibroblasts, to test the dental 

materials. Primary cell lines have a 

predetermined life span and will eventually 

reach a plateau of growth and then die even if 

the conditions for growth are acceptable. 

Because the materials tested would more 

likely come into contact with human 

fibroblasts in vivo, they were chosen to more 

closely represent clinical conditions 33 

This study showed that the 

Epiphany root canal sealer was less toxic than 

AH-26 and Ketac-Endo, the values of the 

cytotoxicity of Epiphany were not significant 

when compared to EndoFill and Apexit. 

These results are in agreement with Souza et 

al34 who showed that Epiphany root canal 

sealer was the only material that presented 

intraosseous biocompatibility compared to 

AH Plus and EndoRez. 

On the contrary, Susini14 et al 

showed that Epiphany + Resilon were the 

most cytotoxic material at 1 and 2 days and 

this toxicity decreases with time until become 

nontoxic at 7 days. They explained this 

toxicity to be due to leaching of uncured 

monomers from the bulk of the resin because 

Epiphany set under anaerobic conditions and 

no curing system leads to a 100% of 

conversion 35. 

The results of this study concerning 

the ZOE sealer were similar to previous 

findings that showed that this material is 

moderately toxic and this toxicity is attributed 

to the release of free eugenol which is the 

main liquid components of ZOE based 

sealers 36, 37. It is interesting to know that 

when zinc oxide eugenolate is formed, most 

of the free eugenol is bound which account 

for the relatively low toxicity of ZOE based 

sealers in the initial setting reaction 38 . 

However, after 48h enough free eugenol 

escaped to cause the reaction to become 

predominantly severe 39 . 

This study indicated that AH-26 

had a moderate to severe cytotoxic reaction 

immediately after mixing and this toxicity 

decreased after 24h. These effects may be due 

to formaldehyde, which is released during the 

initial setting reaction. It has been 

demonstrated that AH-26 is relatively 

38-40 

cytotoxic sealer as it releases 

formaldehyde during and after setting 39 . 

Periapical inflammatory reactions were 

detected when AH-26 was used as sealer 42 . 

Ketac-Endo showed a pronounced 

cytotoxic effect in the early phase of the 

setting reaction and this toxicity decreased 

after 24h. This observation is in agreement 

with a previous study which showed strong 

inflammatory reactions caused by Ketac-Endo 

40 . Schwarze 36 et al. explained this toxicity by 

the high water solubility of the glass ionomer 

cement in the early phase of the setting 

101


reaction leading to the elution of cytotoxic 

substances. 

In conclusion, Epiphany sealer demonstrates 

a clear antibacterial activity, particularly 

against E.faecalis which is the one of the 

most resistant endodontic flora. However it 

had no activity against fungi (C.albicans), so 

the emergence of yeast with resistance to 

Epiphany sealer warrants continued, prudent 

monitoring . 

The cytotoxicity of Epiphany was 

comparable to that of EndoFill and Apexit, on 

the other hand it was less toxic than Ketac- 

Endo and AH-26. 

Further in vivo studies are needed 

to confirm the value of Epiphany sealer using 

a root model. 

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104


دراسه معمليه لتقييم النشاط المضاد للميكروبات و التسمم الخلوي لمادة الابيفاني الغالقة 

لقنوات جذور الأسنان 

امانى بدر-عمرو عبد الرازق-نسرين عمر و فريد بدرية 

هذه الدراسة قيمت 

معمليا النشاط المضاد للميكروبات و التسمم الخلوي لمادة الابيفاني 

الغالقة لقنوات الجذور مقارنة ببعض مواد غالقة أخري معتاد استعمالها و هي الاندوفل،‏ 

ابكسيت ، كتاك-اندو،‏ و 

ايه اتش 

٢٦ في الجزء الأول من هذه الرسالة تم استخدام عدة 

ميكروبات وهم انتيروكوكس فيكالز،‏ 

ستافيلوكوكس ايوريس،‏ 

اشريشيا كولاي،‏ 

و كانديدا البيكان 

لمعرفة التأثير المضاد للميكروبات وذلك باستخدام طريقة انتشار المواد المستخدمة في مادة 

الاجار.‏ 

وقد استخدمت مزرعة من خلايا 

الاربطه المحيطه بالخذر 

الادمي.‏ 

في التقيم الخارجي 

للتسمم الخلوي لمادة الابيفاني وذلك لعد الخلايا الحية بعد حضانة المزرعة مع الابيفاني المخلوط 

حديثا و أخر بعد 

٢٤ ساعة.‏ وقد أسفرت نتائج هذه الدراسة بان مادة الابيفاني لها تاثير فعال في 

قتل البكتيريا وليس 

الكنديدا 

وجد أن التسمم الخلوي لمادة الابيفاني 

متقاربا مع مادة الاندوفل و 

. 

الابكسيت و لكنه اقل سميه من مادتي الكيتاك-اندو و 

٢٦. ايه اتش 

105

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