A Comprehensive in Vitro Comparison of The Biologi

Clinical Oral Investigations (2022) 26:6209–6222
https://doi.org/10.1007/s00784-022-04570-2
ORIGINAL ARTICLE
A comprehensive in vitro comparison of the biological

and physicochemical properties of bioactive root canal sealers
Sabina Noreen Wuersching1 · Christian Diegritz1 · Reinhard Hickel1 · Karin Christine Huth1 · Maximilian Kollmuss1
Received: 28 February 2022 / Accepted: 29 May 2022 / Published online: 3 June 2022
© The Author(s) 2022
Abstract
Objectives To evaluate the biological and physicochemical features of bioactive root canal sealers.
Materials and methods Human periodontal ligament fibroblasts (hPDLF) and human osteoblasts (hOB) were exposed to
eluates of three bioactive root canal sealers, GuttaFlow® bioseal (GF), BioRoot™ RCS (BR), and TotalFill® BC Sealer
(TF), and the epoxy resin–based sealer AH plus® (AH). Cytotoxicity and cellular inflammatory response were evaluated. The
osteogenic potential was examined using human mesenchymal stem cells (hMSC). Film thickness, flowability, and pH were
assessed. Root canal treatment was performed on human extracted teeth to evaluate the sealers’ tightness towards bacterial
penetration. The antibacterial activity against common pathogens in primary root canal infections was tested.
Results AH was severely cytotoxic to hPDLF and hOB (p < 0.001). The bioactive sealers were generally less cytotoxic. IL-6
levels in hPDLF were elevated in the presence of AH (p < 0.05). AH and GF suppressed IL-6 production in hOB (p < 0.05).
AH and BR stimulated the P GE2 production in hPDLF and hOB (p < 0.05). BR was the only sealer that led to calcium deposits
in hMSC (p < 0.05). TF and AH showed the lowest film thickness and the highest flowability. Bacterial tightness was best
in teeth filled with AH and BR. All sealers showed similar antimicrobial activity, but the overall antimicrobial efficacy was
moderate as the bacteria were reduced by just one log scale (p < 0.05).
Conclusions This study revealed favorable in vitro results regarding the biocompatibility of the bioactive root canal sealers.
Clinical relevance Bioactive root canal sealers may be a useful alternative to epoxy resin–based sealers.
Keywords Antimicrobial properties · Apical periodontitis · Bioactivity · Cytotoxicity · Physicochemical properties · Root
canal sealer
Introduction acceptable standards and the root canal filling appears suf-
ficient in the radiograph [3–5]. One of the main causes for
Standard endodontic treatment of teeth with apical periodon- persistent AP are residual microorganisms grouped in bio-
titis (AP) requires a sufficient preparation of the root canal films which have not been entirely eliminated from the root
system to remove the infected pulp tissue, followed by a canal [6]. Despite mechanical instrumentation and thorough
tight apical and coronal seal [1]. The current gold standard disinfection of the pulp cavity, sufficient biofilm removal
for root canal filling materials involves the use of a (semi-) is not always possible due to the complex anatomy of the
solid material such as gutta-percha in combination with a root canal [7]. Particularly the presence of accessory canals,
root canal sealer (RCS) to fill the space between the gutta- ramifications and anastomoses may allow residual bacte-
percha and the root canal wall [2]. It has been reported that ria to persist, because these areas are often inaccessible to
persistent AP occurs in 15–20% of the teeth treated due to mechanical debridement and chemical irrigation solutions
primary AP, even when the endodontic procedure followed [8]. A RCS with additional antimicrobial properties may
therefore not only fill the voids in the radicular dentin
* Sabina Noreen Wuersching
through the pressure applied during obturation with gutta-
[email protected] percha, but, at least in theory, may also inhibit growth of any
residual bacteria enclosed within the root canal system [9].
1
Department of Conservative Dentistry and Periodontology, Among the clinically available RCS, epoxy resin–based
University Hospital, LMU Munich, Goethestrasse 70,
80336 Munich, Germany
sealers such as AH Plus® (AH) are currently the most used.
13
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6210 Clinical Oral Investigations (2022) 26:6209–6222
Various studies have considered AH to be the gold standard (2) osteogenic potential; (3) physicochemical properties
RCS, mainly due to its good dimensional stability, low solu- required for a long-lasting and tight apical seal, such as film
bility, and resistance to resorption [10]. However, several thickness, flow, pH, and tightness towards bacterial penetra-
drawbacks regarding the biocompatibility of AH have been tion; (4) potential antimicrobial properties against common
reported, including cytotoxic effects on fibroblasts, potential endodontic pathogens found in primary root canal infections.
mutagenic activity, and the induction of severe inflammatory
response in bone tissue [11–13]. Concerns have also been
raised that AH may exhibit adverse effects on the adjacent Materials and methods
host tissues and delay the periapical healing of teeth with
AP [14, 15]. Sealers, cells, and bacterial strains
In the recent years, several RCS with supposed bioac-
tive properties have been introduced and since then have Sealers
become the subject of much attention. These novel formula-
tions are claimed to exhibit enhanced biocompatibility and In this study, the bioactive endodontic sealers GuttaFlow®
antimicrobial activity, and even support the regeneration of bioseal (GF), BioRoot™ RCS (BR) and TotalFill® BC
the periapical tissues. BioRoot™ RCS (BR) and TotalFill® Sealer (TF) were examined and compared to AH plus®
BC Sealer (TF) are two calcium silicate–based RCS, whose (AH). The manufacturers and lot numbers of all sealers are
bioactive features are thought to be attributed to their alka- displayed in Table 1.
line pH resulting from calcium hydroxide production and
the release of hydroxide ions in an aqueous environment Cells
[16]. Calcium hydroxide acts antibacterial by damaging
the bacterial membrane and their DNA, but is also known For examining the biological effects of the endodontic seal-
to promote tissue repair by stimulating cell differentiation ers, human periodontal ligament fibroblasts (hPDLF, Lonza,
[17, 18]. These features are beneficial in cases where direct Basel, Switzerland), human osteoblasts (hOB, Promocell,
contact to the surrounding tissues is to be expected, such as Heidelberg, Germany), and human mesenchymal stem
during treatment of AP, root perforations, root fractures, or cells of male Caucasian origin (hMSC, Lonza) were used.
root resorptions. GuttaFlow® bioseal (GF) is a bioceramic hPDLF and hMSC were cultured in high glucose Dulbecco’s
silicone-based RCS which was introduced in an attempt to modified eagle’s medium (DMEM, gibco/life technologies),
combine the filling qualities of gutta-percha and the bio- and osteoblast growth medium (OGM, PromoCell GmbH,
active properties of calcium silicates in one formulation: Heidelberg, Germany) was used for the cultivation of hOB.
fine-grained gutta-percha powder as well as calcium silicate All media were supplemented with 10% fetal bovine serum
particles are incorporated in the silicone matrix of the sealer. (FBS, Merck, Darmstadt, Germany), 100 U/ml of penicillin
Silicon-based RCS have shown several advantages in previ- G and 100 µg/ml streptomycin (Merck). All experiments
ous studies, such as their tight sealing ability, good adapta- were performed with cells at passage 4–8 and all cell types
tion to the root canal wall, and low solubility [19–21]. Root were incubated at 37 °C in a humidified atmosphere contain-
canals can be obturated with GF in a cold paste-only filling ing 5% CO2.
technique without the additional use of gutta-percha points,
and due to its bioactive qualities, it is also considered safe to Bacterial strains
insert large volumes of GF into the root canal system.
All of these biological benefits seem very promising, Five bacterial species were used for testing the sealers’ anti-
but for the bioactive RCS to be a true alternative to epoxy microbial properties: the obligate anaerobes Fusobacterium
resin–based sealers, they must also satisfy the requirements nucleatum (ATCC 49256), Prevotella intermedia (ATCC
concerning their physicochemical properties. Specifically, 25611), Parvimonas micra (ATCC 33270), and Veillonella
low film thickness and high flowability are important fea- parvula (ATCC 17745) as well as the facultative anaer-
tures for a tight seal to prevent bacteria from penetrating the obe Streptococcus oralis (ATCC 35037). All strains were
root canal filling. obtained from the German Collection of Microorganisms
Since the use of bioactive RCS is becoming more popular and Cell Cultures (DSMZ, Braunschweig, Germany) and
among clinicians, there is a need for thorough examinations grown on Schaedler agar plates supplemented with vitamin
of the sealers’ biological and physicochemical properties. K1 and 5% sheep blood (Becton Dickinson, Franklin Lakes,
The main objective of this research is to study the bioac- NJ, USA). Cultivation in liquid medium was performed in
tive RCS GF, BR, and TF compared to the gold standard anaerobic brain–heart-infusion broth (BHI, Becton Dickin-
AH focusing on the following parameters: (1) biocompat- son) supplemented with hemin (5 µg/ml) and vitamin K 1
ibility in terms of cytotoxicity and inflammatory response;
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Clinical Oral Investigations (2022) 26:6209–6222 6211
Table 1 Endodontic sealers used in this study and their chemical composition
Root canal sealer (abbr.) Type Composition References Manufacturer Lot number
AH plus® (AH) Epoxy-amine resin sealerPaste A: bisphenol A/F [11, 45] Dentsply Sirona, York, PA, 1711001109
epoxy resin, calcium USA
tungstate, zirconium
oxide, silica, iron oxide
Paste B: adamantane
amine, N,N-dibenzyl-
5-oxanonane, TCD-
diamine, calcium tung-
state, zirconium oxide,
silica, silicone oil
GuttaFlow® bioseal (GF) Silicone-based gutta-percha Gutta-percha, polydi- [19, 51] Coltene Holding AG, Alt- I67555
with calcium silicate methylsiloxane, zinc stätten, Switzerland
particles oxide, barium sulfate, zir-
conium oxide, platinum
catalyst, color pigments,
micro silver, bioactive
glass ceramic
BioRoot™ RCS (BR) Calcium silicate sealer Liquid: aqueous solution [49, 52] Septodont, Saint-Maur-des- B20650
of calcium chloride and Fossés, France
polycarboxylate
powder: tricalcium silicate,
zirconium oxide and
povidone
TotalFill® BC Sealer (TF) Bioceramic calcium silicate Zirconium oxide, dicalcium [46, 53] FGK Dentaire, La Chaux- 17004SP
sealer silicate, tricalcium sili- de-Fonds, Switzerland
cate, calcium phosphate
monobasic, calcium
hydroxide, fillers
(1 µg/ml). The bacteria were incubated at 37 °C in an anaer- Approximately 5 × 104 cells were seeded into the wells of
obic atmosphere (5% H2, 10% CO2, 85% N2). a 96-well plate an incubated overnight. hPDLF and hOB
cells were treated with the sealer eluates in different con-
Eluate preparation centrations (undiluted, 1:1 dilution, 1:5 dilution in cell cul-
ture medium) as well as with cell culture medium for the
Biological effects of the RCS on hPDLF, hOB, and hMSC control group and incubated for 24 h. CCVK-I was added
were examined using their eluates. Sealers were applied to to the wells according to the manufacturer’s protocol and
the wells of a 24-well plate (200 mg/well) and exposed to the absorbance was measured after 120 min of incubation
UV light for 30 min for sterilization. The sealers were incu- at 450 nm in a spectrophotometer (Varioskan Microplate
bated for 24 h (37 °C, 5% CO2, 95% humidity), allowing Reader, Thermo Fisher Scientific, Waltham, MA, USA).
the material to set. Cell culture medium was added to the Survival rates were computed as a percentage of the control
wells and the well plates were incubated for either 24 h or group and the cytotoxic response was rated as non-cyto-
7 days (37 °C, 5% C O2, 95% humidity). After incubation, toxic (> 90% survival), slight (60–90% survival), moderate
the eluates were sterilized by passing them through a 0.2- (30–60% survival), or severe (< 30% survival) [22].
µm membrane filter (VWR International GmbH, Darmstadt,
Germany) and stored in reaction tubes at − 20 °C until fur- Inflammatory response
ther use.
Interleukin 6 (IL-6) and prostaglandine E2 (PGE2) release
Biocompatibility from hPDLF and hOB was used as a parameter for determin-
ing the cellular inflammatory response to treatment with the
Cytotoxicity sealer eluates. Adherent hPDLF and hOB in 12-well plates
were treated with undiluted sealer eluates for 24 h. Pure
Cytotoxic effects were determined by means of the Col- cell culture medium served as control group. After incu-
orimetric Cell Viability Kit I (CCVK-I, PromoCell) using bation, the IL-6 and PGE2 levels in the supernatants were
the tetrazolium salt WST-8 as an indicator of living cells. measured using a specific enzyme-linked immunosorbent
13

assay (ELISA) according to the manufacturer’s instructions the difference in total thickness of both glass plates with and
(human IL-6 Quantikine-ELISA Kit, R&D Systems, Min- without RCS in between.
neapolis, MN, USA; P GE2 high sensitivity ELISA kit, Enzo
life Sciences, Lörrach, Germany). Standard curves were Flow
generated to calculate the IL-6 and PGE2 concentration. To
exclude interference of the ELISA detection kit with the Sealers were applied to the center of a glass plate (5 mm thick-
RCS eluates, additional standards were performed with each ness). After 180 s, a second glass plate (20 g) was placed on
RCS eluate serving as a diluent. top of the sealer along with a further weight (100 g), measuring
a total weight of 120 g. The maximum and minimum diameter
Osteogenic potential of the compressed sealer between the two glass plates was
measured. If the difference between the two diameters was not
For testing the sealers’ ability to induce osteogenic differen- greater than 1 mm, the mean value was calculated.
tiation in progenitor cells, hMSC at passage 4 were seeded
in the wells of a 6-well plate and incubated overnight. The pH
cells were treated with 7-day RCS eluates in triplicate as
well as with culture medium for the negative control group Polytetrafluoroethylene (PTFE) tubes (1.5 mm inner diam-
and incubated for 7 days. Medium supplemented with eter, 10 mm length) were filled with sealers and left to com-
100 nM dexamethasone, 10 mM β-glycerophosphate, and pletely set. The PTFE tubes were submerged in falcon tubes
50 µM ascorbic acid (all from Merck, Darmstadt, Germany) containing 10 ml of deionized water. The tubes were incu-
served as positive control. The eluates and the medium were bated at 37 °C and pH measurements were performed after
changed every 48 h. The cells were washed three times with 3 h, 6 h, 9 h, 24 h, 3 days, 7 days, 1 month, and 3 months
PBS and fixed with 4% paraformaldehyde solution (Sigma) (827 pH Lab, Deutsche Metrohm GmbH & Co. KG, Filder-
for 30 min. To determine the amount of calcium deposits stradt, Germany).
within the cells, 40 mM alizarin red S staining solution
(ARS, Sigma) was added to each well and incubated for Bacterial penetrability
30 min. After visual examination of the cell cultures, micro-
scopic images of the cells were taken with a phase con- For testing the bacterial penetrability of the endodontic seal-
trast microscope (Axiovert 40 C, Axiocam 305 color, Carl ers, a previously described method was modified [23]. The
Zeiss, Oberkochen, Germany). The ARS was then extracted use of extracted human teeth was approved by the local eth-
from the cells by adding 10% acetic acid and heating the ics committee (registration No. 21–0978 KB). One hundred
cell samples for 10 min at a temperature of 85 °C, followed and four extracted single-rooted, anterior human teeth with
by an incubation on ice for 5 min. After centrifugation for mature apex were selected and x-rayed. The coronal portion
15 min at 20,000 g, the supernatant was neutralized with of all teeth was shortened with a diamond saw to stand-
10% ammonium hydroxide and the absorption at 405 nm ardize the working length (15 mm). Root canal preparation
was determined in a spectrophotometer (Varioskan Micro- was performed with a reciprocating single file (Reciproc
plate Reader). The ARS concentration in the cells was cal- blue R40, VDW, Munich, Germany). Each root canal was
culated using a standard curve with known concentrations irrigated with 10 ml of 3% sodium hypochlorite (NaOCl),
of the dye. 5 ml of 18% ethylenediaminetetraacetic acid (EDTA), and
2 ml of 0.9% sodium chloride. Subsequently, the teeth were
Physicochemical properties sterilized at 121 °C for 15 min. The teeth were divided into
four groups (n = 26) for single cone obturation using the four
Film thickness and flow tests were performed in accordance root canal sealers and gutta-percha. Each filled tooth was
with the DIN EN ISO 6876. placed into an individual reaction tube which was cut off at
the bottom to expose the tooth apex. The interface between
Film thickness the tooth and the reaction tube was sealed with light-curing
flowable resin (SDR flow + , Dentsply Sirona Deutschland
Two flat glass plates with a thickness of 5 mm and a contact GmbH, Bensheim, Germany) and wax. A hole measuring the
area of 225 m m2 were placed on top of each other and the same diameter of the reaction tube was drilled into the cap
total thickness of both plates was measured with a digital of a sterile crimp top vial. The reaction tube with the sealed
micrometer (0.1 µm precision). The same measurement was tooth was placed into the hole, thereby creating two com-
performed with 200 mg of RCS between the glass plates partments with the filled tooth apex as the only interface.
after application of a defined force (150 N) for 10 min. The The experimental set-up is shown in Fig. 1. Methicillin-
film thickness of each RCS was determined by calculating resistant Staphylococcus aureus (MRSA, DSM 11822) was
13

calculated. The experiment was performed in duplicate for

each experimental condition on five individual days.
Statistical analyses
All assessments for the biocompatibility and the physico-

chemical properties were performed in triplicate in inde-
pendent experiments. Statistical analyses were performed
in Python 3.8.0 using scipy and scikit for inferential sta-
tistics and matplotlib for the descriptive analysis [25]. The
Shapiro–Wilk test revealed normal distribution for all data.
Homogeneity of variances was assessed with the Levene’s
test. Data with equal variances were analyzed with a one-
Fig. 1 Diagram showing the experimental setup for examining the way analysis of variances and Tukey’s post hoc test. Com-
bacterial penetrability of the RCS
parisons between groups for data with unequal variances
were performed using Welch’s analysis of variances, fol-
inoculated in BHI medium supplemented with antibiotics lowed by a Tamhane’s T2 post hoc test. P-values < 0.05 were
(10 µg/ml amoxicillin; 10 µg/ml ciprofloxacin) and 500 µl of considered statistically significant. Bacterial penetrability
the MRSA suspension was added to the reaction tubes (upper was analyzed by means of a Kaplan–Meier survival analy-
compartment). The crimp top vial (lower compartment) was sis using the timeframe from the moment the teeth were
filled with clear BHI medium supplemented with antibiotics, incubated in medium until the occurrence of the event of
enough to cover 2 mm of the tooth apex in liquid. This set up interest (turbidity of the medium). No censored observations
was incubated for 28 days at 37 °C in a humidified atmos- occurred during the experiment.
phere containing 5% C O2. Once every 4 days, the bacterial
suspension in the upper compartment was replaced with a
fresh MRSA suspension. The medium in the lower compart- Results
ment was checked for turbidity as a sign of bacterial growth
every day. In case the medium of a specimen turned turbid, Biocompatibility
the day of the event was recorded, and the specimen was not
further incubated. Cytotoxic effects of 24-h and 7-day RCS eluates on hPDLF
and hOB were evaluated with a tetrazolium salt reduc-
Antimicrobial properties tion assay. Cytotoxicity of the RCS in different dilutions
is shown in Fig. 2. Severe toxicity against hPDLF was
For testing the antimicrobial effects of the endodontic seal- observed for undiluted 24-h eluates of AH, GF, and BR
ers, a direct contact test (DCT) was performed by modifying as well as for undiluted 7-day eluates of AH and BR. 1:5
a previously described method [24]. In brief, 200 mg of each dilutions of GF and TF eluates were non-toxic to hPDLF.
sealer were applied to the wells of a 48-well plate and incu- While all undiluted 24-h eluates were severely toxic to
bated at a temperature of 37 °C and a humidity level of 100% hOB, the undiluted 7-day eluates of GF, BR, and TF
for 24 h, allowing the sealers to set. Overnight cultures of F. showed moderate toxicity to hOB. Dilution of the RCS
nucleatum, P. intermedia, P. micra, V. parvula, and S. oralis eluates led to an overall reduced cytotoxicity. All diluted
were diluted to an optical density of 0.1 at 600 nm (Varios- sealers were either moderately, slightly, or non-toxic,
kan Microplate Reader). The five diluted bacterial suspen- except for AH eluates, which were still severely toxic to
sions were combined and 750 µl of the multi-species suspen- hPDLF and hOB in a 1:1 dilution. A 1:5 dilution signifi-
sion were added to each well containing the dried sealer at cantly reduced the cytotoxic effect of all sealers compared
the bottom. Blank wells were used for the control group. The to pure eluates.
well plates were incubated for 48 h in an anaerobic atmos- Cellular inflammatory response to the RCS eluates in
phere. Bacterial growth was observed after 12, 24, and 48 h terms of IL-6 and PGE2-levels is shown in Fig. 3. The 24-h
using a plating and culture method. Colony-forming units AH eluates led to the highest IL-6 levels in hPDLF. All
(CFU) were determined by serially diluting the bacterial sus- other sealer eluates led to similar IL-6 levels as the control
pensions in 0.9% sodium chloride. The diluted suspensions group with medium. P GE2 concentration in hPDLF was
were plated on agar plates and incubated for 48 h. The CFU significantly increased in the presence of 7-day AH elu-
were counted following FDA guidelines (only plates with ates as well as 24-h BR eluates. The 24-h AH eluates and
25 to 250 colonies were considered) and the CFU/ml were 7-day BR eluates also increased P GE2 levels in hPDLF.
13

Fig. 2 Cell survival rate of hPDLF and hOB after exposure to RCS Tamhane’s T2 post hoc analysis. *p < 0.05 compared to the control
eluates of AH, GF, BR, and TF. Survival determined with a colori- group; **p < 0.001 compared to the control group; #p < 0.05 for com-
metric cell viability assay based on the tetrazolium salt WST-8. Data parison between two test groups; ##p < 0.001 for comparison between
shown as the percentage of the control group. P-values determined by two test groups
IL-6 levels in hOB were similar to the control group for after ARS staining, but they showed a similar cell morphol-
all 24-h eluates and for 7-day eluates of BR and TF. In the ogy to the negative control group with cell culture medium.
presence of AH or GF, the IL-6 concentrations in hOB
were significantly reduced compared to the control group. Physicochemical properties
Similar to hPDLF, the PGE2 concentrations in hOB were
significantly enhanced only when incubated with 7-day The results for film thickness and flow are shown in Table 2.
AH and 24-h BR eluates. The film thicknesses of TF, AH, and GF were similar and
fulfilled the requirements of the ISO 6876 standard (film
Osteogenic potential thickness < 50 µm). The film thickness value for BR was
larger than the maximum film thickness required by the ISO
The results for the sealer’s ability to induce osteogenic dif- standard. The film thickness values in increasing order were
ferentiation in hMSC are displayed in Fig. 4 in terms of the TF < AH < GF < BR.
ARS concentration. Only BR led to a significant increase in TF presented the highest flowability among all tested
ARS concentration. Microscopic images of the cells after sealers. GF showed the lowest flowability and was the only
staining with ARS are shown in Fig. 5. The calcium deposits RCS which did not meet the requirements of the ISO stand-
in hMSC (stained red) after incubation with BR are dis- ard (diameter > 17 mm). The diameters indicating the flowa-
tinctly visible. The presence of AH led to total cell death bility in decreasing order were TF > AH > GF > BR.
and caused the cells to detach from the surface. For hMSC Changes in 9 are shown in Fig. 6. For all four sealers, an
incubated with GF or TF, no calcium deposits were observed initial drop in pH within the first 9 h was observed. The pH
13

Fig. 3 Inflammatory response of hPDLF and hOB to AH, GF, BR, post hoc analysis. *p < 0.05 compared to the control group; #p < 0.05
and TF in terms of IL-6 and PGE2 levels. Concentrations [pg/ml] for comparison between two test groups
measured via specific ELISAs. P-values determined by Tamhane’s T2
of BR increased after 24 h and remained stable for the rest

of the experiment, whereas AH, GF, and TF started to show
a slight increase in pH only after 3 months. TF and BR were
the most alkaline for the entire experimental period.
Figure 7 shows the success estimate for the probability of
bacterial penetration of the sealers in a Kaplan–Meier survival
analysis. The overall success rate after an observation period of
28 days was 62% for AH and BR, 58% for GF and 54% for TF.
Antimicrobial efficacy against endodontic

pathogens
Antimicrobial effects of the RCS were assessed in a modified

DCT using a multi-species bacterial suspension with common
endodontic pathogens. Bacterial survival after 12, 24, and 48 h
Fig. 4 Osteogenic differentiation of hMSC after 7 days exposure to exposure to the RCS is shown in Fig. 8. AH was the first to
AH, GF, BR, and TF eluates. Data shown in terms of Alizarin red S show significant antibacterial activity after 12 h, but after 24 h
concentration [mM]. P-values determined by Tamhane’s T2 post hoc of incubation, the presence of all RCS reduced the CFU/ml sig-
analysis. #p < 0.05 for comparison between two test groups nificantly. The mean bacterial growth compared to the control
13

Fig. 5 Microscopic images of hMSC after exposure to different RCS tor of osteogenic differentiation. Arrows indicate possible nodules of
for 7 days (10 × magnification). Cells were stained with Alizarin red mineralization
S staining solution to visualize cellular calcium deposits as an indica-
Table 2 Physical properties of AH GF BR TF

the endodontic sealers used in
this study. Data shown as means Film thickness [µm] 26.33 (± 1.53) 27.33 (± 1.53) 67.67 (± 6.66) 25.00 (± 1.00)
and standard deviation
Flowability [mm] 24.33 (± 1.23) 16.02 (± 0.28) 21.67 (± 1.26) 26.28 (± 1.33)
Fig. 6 pH changes of deionized water containing AH, GF, BR, and TF specimens over a total observation time of 3 months
13

RCS. An epoxy resin–based RCS was used as a comparison,

since it is still considered the gold standard in endodontics.
For evaluating the cytotoxicity of dental materials,
in vitro toxicological examinations are frequently performed
using various cell types, including murine or hamster cells.
Although there are many similarities between human and
animal cells, the number of protein-coding genes can be
very different depending on the species, which may influ-
ence the interpretation of cytotoxic evaluations. Thus, in this
study, we chose to perform all biological examinations with
representative cell types, such as cells of the periodontal
ligament and osteoblasts, both of human origin, assuming
that RCS applied to the root canal of teeth with AP are in
proximity to the periodontal ligament and bone tissue at the
Fig. 7 Kaplan–Meier survival analysis of human extracted teeth filled tooth apex. Furthermore, we aimed to examine initial toxic
with AH, GF, BR, and TF shows the occurrence of bacterial penetra-
tion at each point in days. No censored observations occurred during
effects of the RCS by using eluates extracted after 24 h of
the experiment incubation as well as their late toxic effects after allowing
the components released from the RCS to accumulate for
7 days within the eluates. With these two elution methods
group was 10.46% for AH, 8.21% for GF, 7.27% for BR, and
we were also able to judge the chemical stability of any
17.21% for TF after an exposure time of 48 h.
potentially toxic components released by the RCS. Given
that in vivo RCS eluates are diluted when they are exposed
to tissue fluid and exudate at the tooth apex, the WST-8
reduction assay was performed with RCS eluates both in
Discussion their pure form as well as in different dilutions. Our results
indicate that all four RCS display moderate-to-severe initial
Bioactive RCS are thought to be extremely biocompatible
toxicity to hPDLF and hOB; however, AH was the only RCS
and actively support the regeneration of tissues surrounding
which left no cells to survive after exposure to pure AH elu-
the root canal such as the periapical region and the peri-
ates. A 1:1 dilution reduced the toxicity of all bioactive RCS
odontal ligament. Aside from having these features, it is
to hPDLF and hOB, while AH was still severely toxic to
necessary for an ideal RCS to provide good physical and
both cell types. AH was also found to be generally cytotoxic
chemical properties and show antimicrobial activity against
regardless of the tested cell line. During the osteogenic dif-
endodontic pathogens. This study examined a broad set of
ferentiation experiment, which was performed using hMSC,
clinically relevant features of three bioactive endodontic
we regularly inspected the cells under a light microscope and
Fig. 8 Antimicrobial effects of AH, GF, BR, and TF against a multi- in terms of CFU/ml after an incubation time of 12, 24, and 48 h.
species suspension containing bacterial cultures of F. nucleatum, P. P-values determined by Tukey’s HSD post hoc analysis. *p < 0.05
intermedia, P. micra, V. parvula, and S. oralis in equal amounts (no- compared to the control group; #p < 0.05 for comparison between two
agent control group with BHI medium). Bacterial survival is shown test groups
13

observed that the presence of AH caused the cells to detach a weaker stimulatory effect with 7-day eluates. This find-
from the culture surface after 2 days of incubation. This finding was vice versa for AH, as we observed a more severe
ing marks cell death and also confirms our toxicity results secretion of PGE2 when AH was eluted for 7 days. When
obtained from the WST-8 reduction assay. Furthermore, our interpreting the cytokine release from cells, the total number
results are consistent with data from previous studies which of viable cells capable of producing IL-6 and P GE2 during
have reported severe toxicity of AH against osteoblasts and exposure to the RCS eluates have to be considered. Given
periodontal ligament fibroblasts, while GF, BR, and TF were that the RCS exerted cytotoxic effects as previously ana-
shown to be less cytotoxic than AH [26–30]. As far as dif- lyzed with the WST-8 reduction assay, there were probably
ferences in toxicity of the 24-h and 7-day RCS eluates is also fewer cells secreting cytokines. However, both IL-6 and
concerned, our results revealed an overall lower cytotoxicity PGE2 are stable in vitro, and therefore, the ELISA probably
of the 7-day eluates, especially when diluted. This finding also detected any cytokines which were secreted by the cells
suggests that the toxic components released during the first prior to their death [37, 38]. Another condition affecting
24 h of elution exhibit a relatively low molecular stability, the cellular inflammatory reaction is the mode of cell death
which may have led to a decline in toxic activity of the RCS because the presence of cytoplasmic contents in the extracel-
after only a few days. This would be favorable for all RCS, lular space incites an inflammatory reaction in the remaining
especially since the diluted eluates are more representative cells. However, this circumstance is only to be found in 2
of their long-term cytotoxicity, whereas the undiluted elu- undergoing necrosis, where the membrane integrity is dis-
ates can be viewed as the worst-case scenario because they turbed and cytoplasmic shedding occurs, whereas in apop-
contain all eluted compounds in higher concentrations. totic cell bodies, the cell membrane remains intact, and the
Inflammation as a host reaction to toxic stimuli is char- cytoplasm is retained within the cells [39]. Nonetheless, we
acterized by the release of numerous chemical mediators, cannot draw certain conclusions about the biological inter-
including cytokines, histamines, and prostaglandins. IL-6 is actions solely based on our cell viability and inflammation
an important proinflammatory cytokine which is promptly data, because we are lacking parameters, such as when the
produced by various cell types in response to local tissue cells died or whether they died due to apoptosis or necrosis.
injuries and infections, marking the initial stage of inflam- Furthermore, it is important to bear in mind that in vitro
mation [31]. IL-6 is known to cause an increase in matrix- experiments do not replicate the conditions of cells in an
metalloproteinase-1 (MMP-1), which destroys connective organism, where such biological interactions are far more
tissue by either directly degrading collagen or by activat- complex and several immune cells are present to manage
ing the fibrinolytic protease cascade [32]. Elevated levels the inflammation. This limits the significance of our data to
of IL-6 have also been found to be associated with sympto- predict possible in vivo inflammation caused by the RCS.
matic and larger periapical lesions [33, 34]. PGE2 is a fur- One of the alleged properties of bioactive RCS is their
ther proinflammatory mediator which is synthesized from ability to support the osseous healing of the periapical
arachidonic acid by the cyclooxygenases COX-1 and COX-2 region. Therefore, we tested their osteogenic potential by
and the PGE synthases. The abundance of PGE2 in periapi- incubating hMSC with RCS eluates for 7 days and quantify-
cal tissues is associated with elevated pain sensation caused ing cellular calcium deposits by means of an ARS staining
by PGE2-induced vasodilatation and increased vascular protocol. Calcium content within the cells indicates min-
permeability [33]. Both IL-6 and P GE2 have been identi- eralization and hence the osteogenic differentiation of the
fied as mediators of bone resorption and have been found progenitor cells [40]. BR was the only RCS which led to
to promote resorption of the alveolar bone in patients with nodule-shaped areas that were stained by the ARS stain-
periodontitis by stimulating the osteoclastic activity [35]. ing solution, suggesting signs of possible cellular calcium
However, increased cytokine secretion is not an unfavorable uptake. However, we cannot be certain if these calcium
circumstance by definition. While PGE2 at high concentra- deposits were solely caused by osteogenic differentiation.
tions has been proven to support bone resorption, there is Since BR as well as the other bioactive RCS contain di-
evidence that at low concentrations PGE2 also promotes or tricalcium silicates, it is possible that the stained areas
bone formation by stimulating DNA and collagen synthesis were also due to calcium compounds released from the RCS
in osteoblasts [36]. Our in vitro data show that IL-6 levels which enriched in the cell layers as well. Measuring the cal-
in hPDLF were significantly increased in the presence of cium content as an indicator of osteogenic differentiation
the 24-h AH eluates, while all other eluates led to IL-6 con- may therefore not be the assay of choice when dealing with
centrations similar to those of the control group. However, eluates from materials containing calcium. This limits the
in hOB, the production of IL-6 was reduced by 7-day AH significance of these results, especially in view of the poorly
and GF eluates. AH and BR severely stimulated the P GE2 discernable mineralization in the positive control, where
expression in hPDLF and hOB, but BR led to enhanced the calcium nodules were not stained in a deep red color as
PGE2 production only when eluted for 24 h and displayed expected but merely showed a yellow tint. Therefore, further
13

investigations are necessary to verify the true osteogenic species which was used for inoculating the medium in the
potential of bioactive RCS. upper compartment. The extracted teeth were prepared, dis-
In addition to the cytotoxic and osteogenic effects of the infected, and then obturated in single cone technique using
RCS, this study also addressed their antimicrobial activity gutta-percha and each of the four tested RCS. For block-
in connection with their physicochemical properties. Since ing the dentinal tubes and accessory canals during obtura-
primary root canal infections are typically polymicrobial, tion, low film thickness and high flowability of the RCS are
anaerobic infections, a combination of different bacteria was preferable features. Our evaluations showed that these two
used rather than just one species for testing the antimicro- features were best in AH and TF; however, the first event of
bial effects of the RCS. Previous studies have shown that failure in the bacterial penetrability experiment occurred in
oral streptococci, Fusobacterium spp., Prevotella spp., and the TF group after 5 days of incubation. The lowest overall
P. micra are among the most prevalent species in primary survival rate after 30 days was registered for the TF group
root canal infections [41–43]. Our results revealed that after as well. Initial failure occurred last in the AH group, and
12 h of incubation, AH was the first and only RCS which the best overall survival rate after 30 days was observed for
significantly reduced the number of bacteria in the multi- AH. Despite having a similar film thickness and flowabil-
species suspension. For all other RCS, the first significant ity, the root canal fillings with AH and TF seemed to have
bactericidal activity was recorded after 24 h. After 48 h of varied in tightness. This finding can be explained by further
incubation, the bacterial reduction was similar among all of physical properties that were not part of this research. Data
the tested RCS. No significant difference between the four from a previous study suggest that TF has a higher solubility
groups was observed after 48 h. However, the overall anti- and a lower dimensional stability than AH, which may have
microbial efficacy of all four RCS is to be rated as moderate impaired the tightness of the root canal filling during the
or low, considering that the mean CFU/ml dropped by just 30 days of the experiment [46]. Specifically, TF showed a
0.5–1 log-scales. These findings are also consistent with 7% mass loss after 7 days and a 13% mass loss after 30 days.
data from previous studies, although none of these studies The solubility of AH on the other hand was very low since
assessed the antibacterial effects using multi-species bacte- only a 0.4% mass loss was recorded after 30 days. These
rial suspensions [30, 44, 45]. Furthermore, there is evidence findings are also supported by further studies, which have
from our in vitro results that the antimicrobial efficacy of proved TF to have a low dimensional stability [30, 47].
the RCS may be different depending on the bacterial spe- While BR has also been shown to be more soluble than AH,
cies. Using a culture method for quantifying the number GF is the only RCS which has a reportedly lower solubility
of bacteria allowed us to also distinguish the five bacterial than AH, which could probably be due to its polydimethylsi-
species on the agar plates based on their different morphol- loxane component [48–50]. These results are also consistent
ogy. Thus, we were able to roughly judge to which extent with our observations made for the bacterial penetrability
the growth of each bacterial species was inhibited by the of the RCS tested in this study. Besides initial failure of one
RCS. An example agar plate of the control group showing specimen on day 8, no further specimen in the GF group
the cultured multi-species suspension is displayed as sup- turned turbid until day 23. The GF group therefore showed
plementary information (SI1). In general, S. oralis colonies the longest period in which no event of failure occurred.
were highest in number on all agar plates of the RCS groups, Naturally, the observation time of 30 days presents a limita-
whereas P. intermedia seemed to have barely survived in the tion of this experiment. Considering that a root canal filling
presence of AH, GF, and BR. Notably more P. intermedia ideally remains within the tooth for many years, it would
colonies were visible on the agar plates of the TF group, be interesting to observe the in vitro tightness of root canal
suggesting a lower efficacy of TF against this bacterial spe- fillings in an extended time frame, perhaps until more than
cies. However, to be certain about the specific antibacterial 90% of the specimens have failed.
efficacy against each bacterial species, the DCT would have
to be performed using separate mono-species suspensions
instead of a multi-species mix.
Sufficient tightness of the root canal filling and the coro- Conclusions
nal restoration is a prerequisite for long-term treatment suc-
cess in endodontics [1]. If at least one of both conditions is Within the limitations of an in vitro study, we were able
not provided, there is a higher risk for microleakage through to show favorable results regarding the biocompatibility
the filling material, allowing bacteria to recolonize the root of the tested bioactive RCS GF, BR, and TF. All RCS
canal system and cause persistent infections. We examined exhibited similar antimicrobial properties against common
the tightness of the RCS towards bacterial penetration using endodontic pathogens in primary root canal infections. The
MRSA and a culture medium containing antibiotics to avoid overall antimicrobial efficacy was moderate since none of
cross-contamination and allow bacterial growth only for the the tested RCS achieved a bacterial reduction greater than
13

one log scale. Nonetheless, the bioactive RCS may be a need to obtain permission directly from the copyright holder. To view a
useful alternative to epoxy resin–based RCS, for exam- copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
ple, in cases where the RCS may come into direct contact
with viable cells of the surrounding tissues, such as during
treatment of teeth with AP, for repairing root perforations, References
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Clinical Oral Investigations (2022) 26:6209–6222

A comprehensive in vitro comparison of the biological

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calculated. The experiment was performed in duplicate for

All assessments for the biocompatibility and the physico-

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of BR increased after 24 h and remained stable for the rest

Antimicrobial efficacy against endodontic

Antimicrobial effects of the RCS were assessed in a modified

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Table 2 Physical properties of AH GF BR TF

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RCS. An epoxy resin–based RCS was used as a comparison,

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