Journal of

Clinical Medicine

Article
Microleakage of Restorative Materials Used for Temporization
of Endodontic Access Cavities
Sabina Noreen Wuersching 1 , Luise Moser 1 , Katharina Theresa Obermeier 2 and Maximilian Kollmuss 1, *

1 Department of Conservative Dentistry and Periodontology, LMU University Hospital, LMU Munich,
80336 Munich, Germany; [email protected] (S.N.W.)
2 Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, LMU University Hospital,
LMU Munich, 80337 Munich, Germany; [email protected]
* Correspondence: [email protected]; Tel.: +49-89-4400-59377

Abstract: A tight temporary seal applied to an access cavity is thought to improve endodontic
outcomes. This study aims to assess the bacterial and glucose microleakage of different types and
combinations of temporary restorations. Human-extracted incisors were instrumented, dressed with
a calcium hydroxide paste, and sealed with Cavit W (CW), CW/Ketac Molar (CW/KM), CW/Smart
Dentin Replacement (CW/SDR), Intermediate restorative material/KM (IRM/KM), or Clip F (CF).
Standardized 3D-printed hollow test specimens were manufactured and temporized in the same
manner. The specimens were examined for bacterial and glucose leakage for 28 days. Data were
analyzed using a Kaplan–Meier survival analysis. CW/SDR and CF showed the least bacterial and
glucose leakage over time. CW, CW/KM, and IRM/KM had similarly high levels of glucose leakage,
but CW/KM and IRM/KM provided a tighter seal against bacterial penetration than CW. CW/SDR
and CF should be considered for the sealing of access cavities of teeth previously restored with
methacrylate-based materials.

Keywords: microleakage; bacterial penetration; glucose leakage; temporary restorative materials

Citation: Wuersching, S.N.; Moser, L.;

Obermeier, K.T.; Kollmuss, M.
1. Introduction
Microleakage of Restorative The persistence of bacteria in the root canal is one of the major causes of recurrent
Materials Used for Temporization of endodontic infections [1]. Several steps during endodontic treatment, such as mechanical
Endodontic Access Cavities. J. Clin. instrumentation, antiseptic control with irrigation solutions, the insertion of dressing agents
Med. 2023, 12, 4762. https://doi.org/ with antibacterial properties, and the use of a rubber dam, are intended to eradicate bacteria
10.3390/jcm12144762 from the root canal or prevent bacterial recolonization [2]. A further strategy for improving
Academic Editor: Juan Martin endodontic outcomes is an adequate temporary seal of the access cavity, which prevents
Palomo the contamination of the root canal system with oral fluids and bacteria and forestalls
the seepage of intracanal medicaments between appointments [3]. The significance of
Received: 7 June 2023 a permanent coronal seal after obturation with respect to long-term success has been
Revised: 6 July 2023
previously demonstrated [4]. However, the coronal microleakage of temporary restorations
Accepted: 17 July 2023
must also be considered a potential etiological agent for persistent endodontic infections. In
Published: 18 July 2023
fact, unsatisfactory temporary restorations have been identified as one of the major factors
associated with continuing pain after root canal preparation [5]. Aside from resistance to
microleakage being the main requirement for temporary coronal restorations, there are
Copyright: © 2023 by the authors.
further criteria that should be considered when choosing a temporary restorative material
Licensee MDPI, Basel, Switzerland. for endodontic treatment. From a practical point of view, the material should have a simple
This article is an open access article application procedure, but it should also be easy to remove, leaving virtually no residue
distributed under the terms and in the access cavity. Furthermore, cost effectiveness is an important consideration for
conditions of the Creative Commons clinicians when selecting any dental material for routine use in clinical practice, specifically
Attribution (CC BY) license (https:// in endodontic cases requiring multiple temporary seals.
creativecommons.org/licenses/by/ Among the wide selection of available restorative materials, Cavit has been the most
4.0/). popular temporary material in endodontics for many decades. This popularity is mainly

J. Clin. Med. 2023, 12, 4762. https://doi.org/10.3390/jcm12144762 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2023, 12, 4762 2 of 13

attributed to its simple application procedure, quick removal, and low cost. Cavit is a
eugenol-free calcium sulphate-zinc oxide-based cement that has self-curing properties in
the presence of moisture. The setting reaction of Cavit is initiated by water, which reacts
with calcium sulfate and zinc-oxide-sulfate and thereby produces a hard cement. The
coronal sealing ability of Cavit have been studied previously; however, the in vitro results
seem to vary depending on the method used for assessing the sealing ability [6]. In a recent
systematic review comparing the coronal leakage of different orifice barriers, Cavit showed
superior bacterial microleakage values compared to the other tested materials [7].
To enhance the coronal sealing ability of Cavit, it is a common practice to apply an
additional material to the access cavity on top of a layer of Cavit [8]. For this purpose,
flowable composites or glass ionomer cements (GICs) have been suggested. Both material
classes were primarily developed for restoring class I and II cavities, where a tight marginal
seal is required for the prevention of secondary caries. This makes composites and GICs
attractive for temporization during endodontic treatment. However, composites require an
additional step for a sufficient seal, that is, enamel and dentin conditioning with phosphoric
acid and a dentin-bonding agent. Since composites and GICs originated in restorative
dentistry, they were developed with the premise of being able to last for longer periods of
time, which makes the removal of these materials from an access cavity potentially more
difficult compared to Cavit. Light-curing bulk fill restorative materials such as Clip F allow
for easy placement and removal and should, therefore, be considered for the temporary
sealing of endodontic access cavities. Clip F is a methacrylate-based restorative material
with fluoride-releasing properties that bonds to a tooth without requiring any pretreatment
of the cavity. Its alleged expansion features are thought to ensure a tight seal of a restoration;
however, only few studies have examined the sealing ability of this material so far [9,10].
Another material that is frequently used for temporization in endodontics is IRM
Intermediate restorative material. IRM is a polymethyl methacrylate reinforced zinc oxide-
eugenol cement that was developed for all kinds of intermediate restorations that are
intended to remain in place for up to a year. The eugenol content provides pain relief and is
thus frequently applied to teeth with hypersensitive pulps or as a pulp-capping material in
primary teeth [11]. However, there are conflicting findings from in vitro and in vivo studies
regarding the sealing ability of IRM, with some studies showing equal or more favorable
results than Cavit and other studies demonstrating inferior sealing properties compared to
Cavit [6]. Adding a layer of GIC on top of IRM has been shown to improve the marginal
seal and prevent leakage over a period of up to one month [12].
Although the tightness of some of these materials has been previously examined, there
are only few data on the bacterial leakage of combined restorative materials. Therefore, the
main objective of this study is to assess the sealing ability of different types and combina-
tions of restorative materials used for the temporization of endodontic access cavities.

2. Materials and Methods

2.1. Temporary Filling Materials
Five different combinations of temporary restorative materials were examined in this
study: Cavit W (CW, 3M, St. Paul, MN, USA), Cavit W/Ketac Molar Aplicap (CW/KM,
3M), Cavit W/Smart Dentin Replacement (CW/SDR, Dentsply Sirona, York, PA, USA), IRM
(Dentsply Sirona)/Ketac Molar Aplicap (IRM/KM), and Clip F (CF, Voco Dental GmbH,
Cuxhaven, Germany). Table 1 shows an overview of all materials used in this study along
with the detailed application procedures used for the corresponding restorations.
J. Clin. Med. 2023, 12, 4762 3 of 13

Table 1. Materials, chemical composition, and application procedures of the materials used in
this study.

LOT Application
Material Manufacturer Material Class
Number Procedure
Eugenol-free Apply a 5 mm layer
calcium (3 mm for
3M (St. Paul,
Cavit W (CW) 618059 sulphate-zinc double-layer
MN, USA)
oxide-based restorations) to cavity,
cement add moisture to cavity
Activate capsule for
Ketac Molar Glass ionomer 2 s, mix at 4300 rpm
3M 624122
Aplicap (KM) cement for 10 s, apply a 2 mm
layer to cavity
Apply adhesive
(Scotchbond
Universal) to cavity,
Smart Dentin
Dentsply Sirona Flowable bulk fill air dry for 5 s, light
Replacement 1604000968
(York, PA, USA) composite cure for 20 s, apply a
flow+ (SDR)
3 mm layer of SDR to
cavity, light-cure for
20 s
Intermediate Mix powder and
Zinc oxide
restorative Dentsply Sirona 1606000692 liquid, apply a 3 mm
eugenol cement
material (IRM) layer to cavity
Voco Dental Fluoride-releasing,
Apply a 5 mm thick
GmbH light-curing
Clip F (CF) 1615395 layer to cavity, light
(Cuxhaven, restorative
cure for 40 s
Germany) material

2.2. Bacterial Strains

All bacterial strains were obtained from the German Collection of Microorganisms
and Cell Cultures (DSMZ, Braunschweig, Germany). The following bacteria were used in
this study: Streptococcus mutans (DSM 20523), Aggregatibacter actinomycetemcomitans (DSM
11123), and methicillin-resistant Staphylococcus aureus (MRSA, DSM 11822). All strains were
grown and maintained on Schaedler agar plates supplemented with vitamin K1 and 5%
sheep blood (Becton Dickinson, Franklin Lakes, NJ, USA). For growth in liquid media, the
bacteria were cultured in Brain Heart Infusion Broth (BHI, Becton Dickinson) supplemented
with hemin (5 µg/mL) and vitamin K1 (1 µg/mL). The bacteria were incubated at a
temperature of 37 ◦ C and a humidity level of 60% in a CO2 -enriched atmosphere with
5.8% CO2 .

2.3. Determination of Antibiotic Susceptibility Profiles

The bacterial penetration experiment required a BHI medium supplemented with
antibiotics to avoid cross-contamination and prevent growth of typical pathogens that
may originate from extracted teeth. The antibiotic susceptibility of S. mutans and A. actino-
mycetemcomitans to ampicillin (AMP) and ciprofloxacin (CIP) was determined using the
ETEST method (bioMèrieux SA, Marcy-l’Étoile, France). In brief, colonies of S. mutans,
A. actinomycetemcomitans and MRSA were inoculated in BHI and adjusted to a McFarland
Standard of 0.5. The bacterial suspensions were evenly streaked onto individual Schaedler
agar plates using sterile cotton swabs. The agar plates were incubated with ETEST strips un-
der the required growth conditions for 24 h, and minimum inhibitory concentrations (MICs)
were read and interpreted according to the breakpoints reported in the guidelines from
the Clinical and Laboratory Standards Institute (CLSI) [13,14]. All tests were performed
in triplicate for each bacterial strain. Based on the MICs, antibiotic concentrations were
selected in order to supplement the BHI medium with AMP and CIP. The prepared BHI
J. Clin. Med. 2023, 12, 4762 4 of 13

was examined in terms of the selective bacterial growth of MRSA by plating and culturing
bacterial suspensions of all three strains grown in BHI supplemented with the antibiotics.

2.4. Preparation of Human-Extracted Teeth

To test the tightness of the temporary restorative materials, a previously described
method was used and modified [15,16]. The use of extracted human teeth was approved
by the local ethics committee (registration No. 23-0431 KB). A total of 75 extracted, single-
rooted, anterior human teeth with mature apices and no signs of caries in the root area
were selected and X-rayed. The coronal portion of all teeth was shortened with a diamond
saw to standardize the teeth to a working length of 16 mm. The access opening of each
tooth was also standardized (5 mm depth, 2.5 mm diameter). The coronal third of the
root canal was flared with Gates–Glidden burs, and root canal preparation was performed
with a reciprocating single file (Reciproc blue R40, VDW, Munich, Germany). Each root
canal was irrigated with 15 mL of 3% sodium hypochlorite (NaOCl) and 5 mL of 20%
ethylenediaminetetraacetic acid (EDTA), and the teeth were then sterilized at 121 ◦ C for
15 min. The apical 7 mm sections of the root canal were filled with a calcium hydroxide
paste (Ultracal XS, Ultradent Products, South Jordan, UT, USA) using a 28 G (0.35 mm)
needle (VMK Endoneedle, VMK, Dilbeek, Belgium). The teeth were divided into five
experimental groups (n = 15) to seal the opening of the access with the different restorative
materials. Detailed specifications of the filling procedure for each material are shown
in Table 1.

2.5. Preparation of 3D-Printed Hollow Test Specimens

Since the inherent anatomical variations of the extracted teeth can affect the corre-
sponding results, standardized test specimens that simulate an access cavity in a resin-based
material were created. A hollow, conical cylinder measuring 8 mm in height and with
an inner diameter of 2.5 mm at the frustum base corresponding to the dimensions of the
access openings of the extracted teeth was virtually designed using CAD software (Tizian
Creativ RT-Software, Version 3.1, Schütz Dental GmbH, Rosbach, Germany). A diagram
showing the dimensions of the conical cylinder is displayed in the Supplementary Material
(Figure S1). The template was exported as a standard tessellation language (stl) file and sent
to a 3D-printer (Form 2, Formlabs Inc., Somerville, MA, USA), where 150 specimens were
produced from a printable resin material (Dental LT Clear Resin V2, Formlabs). The resin
specimens were post-processed according to the manufacturer’s instructions by washing
them in isopropanol for 20 min (Form Wash, Formlabs) and post-curing them at 60 ◦ C for
60 min (Form Cure, Formlabs). The hollow test specimens were filled with the restorative
materials in the same manner as described in Table 1.

2.6. Bacterial Penetration

Each filled tooth and hollow test specimen was placed into an individual reaction tube
that was cut off at the bottom to expose the tooth apex/small frustum base. The interface
between the tooth/test specimen and the reaction tube was sealed with light-curing flow-
able resin (SDR flow+, Dentsply Sirona Deutschland GmbH, Bensheim, Germany) and
wax. A hole measuring the same diameter of the reaction tube was drilled into the cap of a
sterile crimp top vial. The reaction tube with the sealed specimen was placed into the hole,
thereby creating two compartments with the filled tooth apex/hollow test specimen as the
only interface. The experimental setup is shown in Figure 1a. MRSA was inoculated in BHI
medium supplemented with antibiotics (10 µg/mL AMP; 10 µg/mL CIP), and 500 µL of
the MRSA suspension was added to the reaction tubes (upper compartment). The crimp top
vial (lower compartment) was filled with clear BHI medium supplemented with antibiotics,
enough to cover 2 mm of the tooth apex/hollow test specimen in liquid. This setup was
incubated for 28 days at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Once every
four days, the bacterial suspension in the upper compartment was replaced with a fresh
MRSA suspension. The medium in the lower compartment was examined for turbidity, as
J. Clin. Med. 2023, 12, 4762 5 of 13

liquid. This setup was incubated for 28 days at 37 °C in a humidified atmosphere contain-
J. Clin. Med. 2023, 12, 4762 5 of 13
ing 5% CO2. Once every four days, the bacterial suspension in the upper compartment
was replaced with a fresh MRSA suspension. The medium in the lower compartment was
examined for turbidity, as a sign of bacterial growth, every day. In case the medium of a
aspecimen
sign of bacterial
turned growth, every
turbid, the dayday. In case
of the thewas
event medium of a specimen
recorded, turned turbid,
and the specimen was the
not
day of the event was recorded, and the specimen was not further incubated.
further incubated. The medium in the lower compartment was then examined for the The medium
in the lower
presence compartment
of MRSA by platingwas
thethen examined
medium for theagar
on selective presence
platesoffor
MRSA
MRSA bygrowth
plating(BBL
the
medium on selective agar plates for MRSA growth (BBL CHROMagar ® MRSA, BD). MRSA
CHROMagar MRSA, BD). MRSA produces typical pink colonies resulting from hydrol-
®
produces
ysis of thetypical pink colonies
chromogens added toresulting from
the agar, hydrolysis
while coloniesof the other
from chromogens
bacterialadded to the
species ap-
agar, while colonies from other bacterial species appear white. Success of
pear white. Success of each material group was analyzed via Kaplan–Meier-survival anal- each material
group was
ysis, and analyzed
the averagevia Kaplan–Meier-survival
probability analysis,
of success (event-free time)and thedetermined
was average probability
by calculat-of
success (event-free time) was determined by calculating
ing the area under the curve (AUC) for each material group. the area under the curve (AUC)
for each material group.

(a) (b)
25G x 1 1/2” cannula

Crimp top vial

Reaction tube

Upper compartment (MRSA suspension)

Upper compartment (1M D-glucose solution)

Tooth or 3D-printed resin form

with temporary restoration

Interface sealed with resin and wax

7 mm calcium hydroxide paste
Lower compartment (BHI suppl. with AMP/CIP)
Lower compartment (0.2% NaN3 solution)

Figure 1. Diagram
Figure Diagramshowing
showingthe
theexperimental
experimentalsetup forfor
setup assessing (a)(a)
assessing bacterial penetration
bacterial andand
penetration (b)
glucose
(b) leakage.
glucose leakage.

2.7.
2.7. Quantitative
Quantitative Microleakage
Microleakage
Quantitative
Quantitative analysis
analysis of
of microleakage
microleakage through
through thethe temporary
temporary fillingfilling materials
materials was
was
performed using the glucose leakage method by modifying a previously
performed using the glucose leakage method by modifying a previously described proto- described proto-
col
col [17,18].
[17,18]. Seventy-five
Seventy-fivehollow
hollowtest
testspecimens
specimenswerewereprepared
prepared andandfilled with
filled thethe
with temporary
tempo-
filling
rary filling materials as described above. A setup similar to that employed for the bacterial
materials as described above. A setup similar to that employed for the bacterial
penetration
penetration experiment
experiment was was used,
used, involving
involving aa crimp
crimp top
top vial
vial as
as aa lower
lower compartment
compartment and and
aa reaction
reaction tube as an upper compartment, with the hollow test specimen containing
tube as an upper compartment, with the hollow test specimen containing thethe
temporary
temporary filling
filling materials
materials serving
serving as
as the
the only
only interface.
interface. AA total
total ofof 3.5
3.5 mL
mL ofof water
water was
was
added
added to to the
the lower
lower compartment,
compartment, andand 11 M
M D-glucose
D-glucose solution
solution waswas added
added to to the
the upper
upper
compartment as the tracer substance. Both the water and glucose contained 0.2% sodium
compartment as the tracer substance. Both the water and glucose contained 0.2% sodium
azide to prevent bacterial growth. A sterile 25 G × 1 1/2” (0.5 mm × 40 mm) cannula
azide to prevent bacterial growth. A sterile 25 G × 1 1/2” (0.5 mm × 40 mm) cannula
(B.Braun, Melsungen, Germany) was placed in the lower compartment of each specimen
(B.Braun, Melsungen, Germany) was placed in the lower compartment of each specimen
to regularly withdraw liquid with a syringe for measurement. The experimental setup is
to regularly withdraw liquid with a syringe for measurement. The experimental setup is
shown in Figure 1b. The specimens were incubated at 37 ◦ C and 100% humidity and in 5.8%
shown in Figure 1b. The specimens were incubated at 37 °C and 100% humidity and in
CO2 for 28 days. Glucose leakage through the temporary filling materials was assessed
every 2 days by quantifying the glucose concentration in the lower compartment via an
enzymatic assay (Glucose (HK) Assay Kit, Merck, Darmstadt, Germany). The Glucose (HK)
Assay Reagent was reconstituted according to the manufacturer’s instructions, and 200 µL
of the test solution was removed from the lower compartment. A total of 100 µL of the
J. Clin. Med. 2023, 12, 4762 6 of 13

detection reagent and 10 µL of the test solution were combined in the wells of a 96-well
plate and incubated for 15 min at 37 ◦ C. Absorbance was measured at 340 nm using a
spectrophotometer (Varioskan Microplate Reader, Thermo Fisher Scientific, Waltham, MA,
USA). To convert the optical densities (OD) to glucose concentrations, a standard curve
describing the relationship between OD and predetermined glucose concentrations was
generated on each day of measurement.

2.8. Statistical Analyses

All statistical evaluations were performed in Python 3.8.0 using the packages pandas,
sklearn, and lifelines for inferential statistics and matplotlib for the descriptive analyses [19].
To analyze the probability of success of each material group, a Kaplan–Meier-survival anal-
ysis was conducted using the timeframe from the moment the specimens were incubated
until the event of interest (i.e., the point at which the medium became turbid) occurred.
There were no censored observations during the experiment.

3. Results
3.1. Minimum Inhibitory Concentrations
The MICs of AMP and CIP for each strain are summarized in Table 2. Both S. mutans
and A. actinomycetemcomitans were susceptible to AMP and CIP according to the breakpoints
reported in the CLSI guidelines. MRSA was resistant to AMP and CIP and showed
no inhibition zone with respect to either antibiotic. Sample images of agar plates after
incubation with the ETEST strips are shown in the Supplementary Material (Figure S2).
Based on these results, a concentration of 10 µg/mL was chosen as the amount at which
both AMP and CIP would be added to the BHI. No bacterial growth of S. mutans and
A. actinomycetemcomitans was observed after plating and culturing the suspensions grown
in AMP/CIP-BHI, and there was no difference in the bacterial growth of the MRSA strains
compared to antibiotic-free BHI.

Table 2. Minimum inhibitory concentrations (MIC) of bacterial strains against Ampicillin and
Ciprofloxaicin determined using the ETEST method. MICs interpreted according to guidelines of the
Clinical and Laboratory Standards Institute (CLSI). R, resistant; S, susceptible.

Strain Antibiotic Range MIC (µg/mL) Interpretation

Ampicillin 0.016–256 0.094 S
S. mutans
Ciprofloxacin 0.002–32 1.00 S
Ampicillin 0.016–256 1.00 S
A. actinomycetemcomitans
Ciprofloxacin 0.002–32 0.004 S
Ampicillin 0.016–256 >256 R
MRSA
Ciprofloxacin 0.002–32 >32 R

3.2. Bacterial Penetration

Figure 2 shows the Kaplan–Meier survival analysis of the teeth (a) and hollow test
specimens (b) filled with the different restorative materials. The average probability of
success (event-free time), which was determined by calculating the area under the curve
(AUC), and the percentage of event-free specimens after 14 and 28 days of incubation are
shown in Table 3 for each material group. In both experimental setups, the highest average
event-free time was recorded in the CW/SDR and CF groups, whereas CW alone had the
smallest AUC. The largest differences between the AUC obtained from the setup with the
hollow test specimens and the setup with teeth was observed for the CW/SDR and CF
groups. CW/KM and IRM/KM showed a similar success rate in both experimental setups.
After 14 days of incubation, CW/SDR and CF had the highest percentages of event-free
specimens in both experimental setups. At the end of the observation period (28 days), the
highest number of tight specimens was observed in the specimens sealed with CW/SDR in
both setups.
 hollow test specimens and the setup with teeth was observed for the CW/SDR and CF
groups. CW/KM and IRM/KM showed a similar success rate in both experimental setups.
After 14 days of incubation, CW/SDR and CF had the highest percentages of event-free
J. Clin. Med. 2023, 12, 4762
specimens in both experimental setups. At the end of the observation period (28 days), the
7 of 13
highest number of tight specimens was observed in the specimens sealed with CW/SDR
in both setups.

Figure 2.
Figure 2. Kaplan–Meier
Kaplan–Meiersurvival
survivalanalysis indicating
analysis thethe
indicating occurrence of bacterial
occurrence penetration
of bacterial in tem-
penetration in
porized (a) human-extracted teeth and (b) 3D-printed hollow test specimens. No observations
temporized (a) human-extracted teeth and (b) 3D-printed hollow test specimens. No observations were
censored during the experiment.
were censored during the experiment.

Table 3. Area under the Kaplan–Meier curve (AUC) indicating the average probability of success
Table 3. Area
(event-free under
time) the Kaplan–Meier
for each material groupcurve
along(AUC) indicating
with the the of
percentage average probability
event-free of success
specimens after 14
(event-free time) for each material group along with the percentage of event-free specimens
and 28 days of incubation. All values are shown for the experimental setups with extracted after 14
teeth
and
and28 days of
hollow testincubation.
specimens.All values are shown for the experimental setups with extracted teeth and
hollow test specimens.
Cavit W + Cavit W + IRM + Ketac
Cavit W Clip F
Cavit W
Ketac Molar
Cavit W+ SDRW +
Cavit Molar
IRM + Ketac
Clip F
AUC Teeth 6.20 Ketac Molar
14.00 SDR
17.20 Molar
14.30 16.40
AUC Teeth
Success after 6.20 14.00 17.20 14.30 16.40
6.67 46.67 66.67 53.33 66.67
14 days after
Success (%) 6.67 46.67 66.67 53.33 66.67
14 days (%)
Success after
Success after 6.67
6.67 20.00
20.00 40.00
40.00 33.33
33.33 26.67
26.67
28
28days
days (%)
(%)
AUC Hollow
AUC Hollow 4.93
4.93 13.17
13.17 26.80
26.80 13.07
13.07 26.07
26.07
test
testspecimens
specimens
Success after
Success after 6.67
6.67 33.33
33.33 100.00
100.00 33.33
33.33 100.00
100.00
14 days (%)
14 days (%)
Success after
Success 0.00 33.33 93.33 13.33 86.67
28 daysafter
(%) 0.00 33.33 93.33 13.33 86.67
28 days (%)
3.3. Glucose Leakage
3.3. Glucose Leakage
The results of the glucose leakage experiment are shown in Figure 3. In the CW,
The results of the glucose leakage experiment are shown in Figure 3. In the CW,
CW/KM, and IRM/KM groups, the glucose concentration in the lower compartment
CW/KM, and IRM/KM
increased rapidly withingroups,
the first the
fiveglucose concentration
days. After 10 days of in the lowerthe
incubation, compartment in-
glucose levels
creased
in rapidly
the lower within the first
compartment werefive days. After
saturated 10 days
in these of incubation,
three groups. The thelowest
glucosevalues
levels of
in
the lower compartment were saturated in these three groups. The lowest values
glucose leakage were observed in the specimens sealed with CF and CW/SDR, where the of glucose
leakagelevels
glucose were observed in the
in the lower specimensdid
compartment sealed with CFconcentrations
not exceed and CW/SDR,ofwhere the glucose
0.07 mg/mL and
levels in the lower compartment
0.18 mg/mL, respectively. did not exceed concentrations of 0.07 mg/mL and 0.18
mg/mL, respectively.
J. Clin.
J. Clin. Med.
Med. 2023, 12, 4762
2023, 12, 4762 8 8of
of 13
13

Figure
Figure 3.
3. Glucose
Glucose microleakage
microleakage of
of temporized 3D-printed hollow
temporized 3D-printed hollow test
test specimens.
specimens.

4. Discussion
Temporary restorations
restorations are a potential weak spot of root canal treatments because
bacteria and oral
bacteria and oral fluidsfluids maymay penetrate
penetrate along
along thethe margins
margins of of seemingly
seemingly intact
intact restorations,
restorations,
leaving clinicians
leaving cliniciansuncertain
uncertainabout aboutthe sterility
the sterilityof of
thethe
pulp chamber
pulp chamberandandthe root canals
the root after
canals
temporization.
after temporization.To abolish the need
To abolish for dressing
the need for dressingagents and and
agents a temporary
a temporary seal,seal,
one-visit
one-
treatments
visit havehave
treatments been been suggested, particularly
suggested, particularlyfor clinical casescases
for clinical with with
necrotic pulpspulps
necrotic or vital
or
teeth with clinical signs of inflammation [20]. Although one-visit
vital teeth with clinical signs of inflammation [20]. Although one-visit treatments have treatments have been
proven
been to be to
proven just
beasjusteffective as multi-visit
as effective treatments
as multi-visit in terms
treatments of clinical
in terms success,
of clinical they
success,
are associated with a higher risk of postoperative pain or flare-ups
they are associated with a higher risk of postoperative pain or flare-ups [20,21]. Therefore, [20,21]. Therefore,
aa multi-visit
multi-visittreatment
treatmentprotocol
protocolis is
thethe preferable
preferable alternative
alternative for for infected
infected teethteeth causing
causing pre-
preoperative pain in order to reduce the chances for postoperative
operative pain in order to reduce the chances for postoperative complications as well as complications as well
as the
the unnecessary
unnecessary or excessive
or excessive usenonsteroidal
use of of nonsteroidal anti-inflammatory
anti-inflammatory drugs, drugs,
whichwhich
are com-are
commonly used to treat postoperative endodontic
monly used to treat postoperative endodontic pain [20,22,23]. pain [20,22,23].
During multi-visit treatments, a sufficient temporary coronal seal is a prerequisite
During multi-visit treatments, a sufficient temporary coronal seal is a prerequisite for
for avoiding the contamination of the root canal system with oral fluids and bacteria.
avoiding the contamination of the root canal system with oral fluids and bacteria. The
The presence of bacteria in the root canal at the time of filling is considered a risk factor
presence of bacteria in the root canal at the time of filling is considered a risk factor for
for developing posttreatment apical periodontitis [24]. Ideally, the root canal system
developing posttreatment apical periodontitis [24]. Ideally, the root canal system will be
will be thoroughly disinfected at the final appointment to remove any residual bacteria.
thoroughly disinfected at the final appointment to remove any residual bacteria. None-
Nonetheless, leakage through the temporary restoration may facilitate entry of Enterococcus
theless, leakage through the temporary restoration may facilitate entry of Enterococcus fae‐
faecalis, a bacterial species that is typically found in teeth in need of endodontic retreatment
calis, a bacterial species that is typically found in teeth in need of endodontic retreatment
and, less frequently, in primary endodontic infections [25–27]. The problem with this
and, less frequently, in primary endodontic infections [25–27]. The problem with this spe-
species is that it is able to withstand conventional eradication measures because it is
cies is that it is able to withstand conventional eradication measures because it is equipped
equipped with certain survival and virulence factors, such as the ability to invade dentinal
with certain survival and virulence factors, such as the ability to invade dentinal tubules,
tubules, survive extremely alkaline environments, and resist nutritional deprivation [28]. E.
survive extremely
faecalis and severalalkaline environments,
other bacteria found inand resist nutritional
persistent infectionsdeprivation [28]. E. faecalis
have been demonstrated
and several other bacteria found in persistent infections have
to be resistant to calcium hydroxide dressing agents, which highlights the necessity been demonstrated to be
of
resistant
avoidingto calcium
the hydroxide
colonization of thedressing
root canalagents,
systemwhich highlights
with the necessity
such bacteria by ensuringof avoiding
a tight
the colonization
coronal seal [29]. of the root canal system with such bacteria by ensuring a tight coronal
seal [29].
In this study, bacterial and glucose leakage was assessed over an observation period
of 28In thisto
days study,
judgebacterial
the sealing and ability
glucoseofleakage was assessed
the temporary fillingover an observation
materials period
as a function of
of
time. We employed two methods to address different factors relating to the tightness of
28 days to judge the sealing ability of the temporary filling materials as a function of
time. We employed
the restorations. Whiletwo bacterial
methods penetration
to address different
seems the factors
most relating
plausible toandthe tightness
biologicallyof
the restorations.
relevant method While
for ourbacterial
researchpenetration
question, it seemsdoes not theaccount
most plausible
for voidsand thatbiologically
are smaller
relevant
than the average size of a bacterial cell (0.5–1.0 µm). However, these gaps areare
method for our research question, it does not account for voids that smaller
of interest
than the average size of a bacterial cell (0.5–1.0 µm). However,
from a clinical point of view because they may provide pathways for smaller molecules, these gaps are of interest
from
such asa clinical point
toxins and of view
other because
bacterial they may
products, provide
to travel to thepathways
periapexfor smaller
[30]. Severalmolecules,
methods
such as toxinsmicroleakage
for assessing and other bacterial
have beenproducts,
previouslyto travel to the periapex
described. Most methods[30]. Several
are basedmethods
on the
for
sameassessing
principle,microleakage
that is, detecting haveabeen
tracerpreviously described.
upon penetration along Most
the methods
margins of arethe
based on
coronal
the same principle, that is, detecting a tracer upon penetration along the margins of the
J. Clin. Med. 2023, 12, 4762 9 of 13

restoration or the obturated canal of an extracted tooth. The most frequently used method is
dye penetration with dyes such as methylene blue, basic fuchsin, or India ink. However, the
problem with this method is that it leads to a great deal of variation in the results because
it is usually associated with assigning a score using a scoring system, which leads to a
subjective interpretation of the degree of leakage [31]. Furthermore, it has been reported
that dye penetration can lead to misleading conclusions due to alterations of the properties
of the dye depending on the pH or the presence of ions [32]. The use of radioactive isotopes
as tracers allows for better quantification of the microleakage via the placement of a known
quantity of an isotope into the pulp chamber of an extracted tooth and allowing the tracer to
leak into an outside medium, where the amount of the radioactive isotope can be measured
using a scintillation counter [33]. However, the downsides of this method are that it is
extremely technique-sensitive and has questionable clinical relevance in addition to posing
a risk due to the exposure to potentially hazardous radiation [31].
The glucose leakage model modified according to Xu et al. allows for a nondestructive
quantification of microleakage over time without suffering from the shortcomings of the
methods mentioned above [17]. Glucose seemed to be a suitable tracer for our research
question, not only because it serves as an indicator of smaller gaps within a restoration
due to its small molecular size (180 Da) but also because glucose serves as a nutrient for
bacteria, which, once it has penetrated through the temporary restoration, may promote the
growth of residual bacteria colonizing the root canal. The advantages of this method are
its great sensitivity, ease of operation, and comparatively low costs. The fact that glucose
is relatively stable in vitro is a further advantage of the glucose penetration method. This
feature allows us to determine microleakage continuously, whereas other methods such
as dye penetration require an end-point for assessing the penetration depth along the
root canal.
Previous studies employing bacteria as a tracer for leakage used species that are often
found in infected root canals, such as Enterococcus faecalis, Streptococcus mutans, or Streptococ-
cus gordonii [8,12,34]. Using bacteria in accordance with their clinical relevance seems to be
a viable approach; however, this protocol is fraught with difficulties in controlling the bac-
terial populations due to potential cross-contamination and other bacteria originating from
the extracted teeth. To overcome this common limitation, we modified the methodology
in our study and used MRSA as a microbial tracer in combination with a culture medium
containing antibiotics. One may now question the clinical relevance of this approach, as
MRSA is not a typical species found in root canal biofilms [35]. However, this concept is
based on the premise that bacteria do not significantly vary in size and that our goal is to
make a dichotomous decision on whether bacterial cells were able to penetrate along the
margins of the restoration. The advantage of this method is that it allows for the selective
growth of the tracer bacteria while avoiding contamination with other bacterial species. To
determine suitable antibiotic concentrations for a BHI medium with efficient antibacterial
activity against Gram-positive and Gram-negative bacteria, S. mutans and A. actinomycetem-
comitans were used. Aside from these two species, which served as representative bacteria
for laboratory cross-contamination, S. mutans and A. actinomycetemcomitans were chosen
because they are typical species found in teeth affected by the most common oral diseases,
i.e., caries and periodontitis. In addition, sterility was double-checked upon the occurrence
of an event by plating the medium from the lower compartment on chromogenic agar
plates that allow for the selective identification of MRSA growth.
The results from the bacterial leakage experiments in conjunction with those from
the glucose penetration experiment may reflect the sealing ability of the tested materials
in a clinical context. Our proposition that the presence of nutrients in the root canal and
bacteria may be correlated was confirmed by the fact that the specimens with a high degree
of glucose leakage also had a high degree of bacterial leakage. This was the case for the
access cavities sealed with CW, CW/KM, or IRM/KM. In the CW and CW/KM groups,
we observed a drop in glucose concentrations between days 5 and 7. This reduction might
have been to possible interactions between glucose and components within the restorative
J. Clin. Med. 2023, 12, 4762 10 of 13

materials. Previous research has demonstrated that certain endodontic filling materials,
especially those containing calcium hydroxide, react with glucose to form gluconic acid,
thereby reducing the glucose concentration. A further compound the authors tested with
regard to its reactivity with glucose was calcium sulphate, which also led to a slight
reduction in glucose content, but the difference in concentrations was not significant [36].
Based on this information, calcium sulphate, one of the main components of Cavit, may
have caused the glucose concentration to slightly drop in the CW and CW/KM specimens,
especially since these restorations showed early signs of microleakage, allowing for the
glucose in the lower compartment to interact with the CW portion. Furthermore, the
authors of the previously mentioned study had chosen a period of 7 days for the immersion
of the specimens in the glucose solution, which happens to be the same time until the
glucose concentration dropped in the presence of the CW and CW/KM specimens.
Based on the findings obtained from several studies examining the adhesive bond
to the tooth, it is not surprising that specimens sealed with CW/SDR were superior in
terms of tightness [37]. The fact that no drop in glucose concentration occurred in the
CW/SDR specimens supports this proposition, as the glucose probably never reached the
CW portion of CW/SDR in significant concentrations. Access cavities that were sealed
solely with CW presented the highest levels of bacterial leakage, whereas CW with a layer
of KM on top seemed to provide a slightly improved seal against bacterial leakage but
not against glucose microleakage. IRM/KM and CW/KM presented similar results in
terms of bacterial and glucose leakage; however, the onset of glucose leakage from the
IRM/KM specimens occurred later than that from the CW/KM specimens. These findings
are in accordance with previous studies that demonstrated an equal or better seal using
IRM than that provided by Cavit against bacterial penetration [12,38,39]. We obtained
promising results for CF, a light-curing methacrylate-based material, which has not yet
been as widely studied as Cavit and IRM. Previous studies that assessed the marginal
tightness of different restorative materials with dye penetration (fluorescent silver staining
and methylene blue) demonstrated an equal or superior seal using CF compared to that
using IRM or Cavit G [10,40]. A further study examined the sealing ability of temporary
restorations in previously restored teeth (for which dye penetration with basic fuchsine
was employed) and found that Clip, the forerunner material of CF, provided the best
seal against microleakage not only at amalgam and composite interfaces but also at the
tooth–tissue interface [41]. The only other in vitro study employing bacteria as a tracer
for coronal leakage reported a 50% chance of success for teeth sealed with Clip after an
incubation time of 30 days with a S. mutans bacterial suspension [42]. The good sealing
ability of CF may be attributed to its expansion properties reported by the manufacturer,
which have not yet been confirmed through independent research. The product data sheet
for CF indicates a 0.5% level of water sorption after immersion in water for 14 days, which
could have contributed to the tight seal within the access cavities. Moreover, it should
be noted that CF, a material with no known mechanism of adhesive bonding to tooth
tissues, has been reported to provide an excellent bond with other methacrylate-based
materials [41,43]. This feature must be considered when interpreting the results from
our experiment with the 3D-printed hollow test specimens to avoid an overestimation
of the material’s sealing ability. Even without pretreatment of the hollow test specimens,
the unpolymerized methacrylates in CF might have created a chemical bond with the
methacrylates in the resin, which may explain the low occurrence of events in the setup
with the 3D-printed test specimens. This notion also applies to the 3D-printed specimens
sealed with CW/SDR, which was used along with a universal adhesive capable of creating
a chemical bond with the resin’s surface. In contrast, the properties of the 3D-printed forms
might have led to an underestimation of the tightness of CW/KM and IRM/KM because
KM is a GIC, which is primarily designed to bond to tooth tissues and not methacrylates.
On the other hand, CW and IRM do not have an adhesive bonding mechanism. Instead,
their sealing ability is based on hygroscopic expansion, which produces a seal within the
cavity [44]. It is unlikely that the sealing ability of CW and IRM was impaired by the
J. Clin. Med. 2023, 12, 4762 11 of 13

use of the 3D-printed hollow test specimens, especially since a similar survival curve was
obtained for both experimental setups. However, it should be noted that teeth in need of
root canal treatment often have coronal restorations that are not always removed prior to
gaining access to the pulp chamber. This may significantly affect the sealing properties of
the materials depending on the type of material used for the coronal restoration. Therefore,
CF may be an option when sealing the access cavities of teeth with coronal composite
restorations, or for methacrylate-based materials in general. From a practical point of view,
a disadvantage of using materials with a dentin-bonding agent is that they are harder to
remove than cements such as CW or IRM. As re-entry through composite restorations
requires the use of diamond burs, there is a higher risk of weakening the tooth when
attempting to remove all adhesive residues potentially blocking the dentin tubules of the
pulp chamber wall. The double-layered restorations involving a coronal layer of composite
or GIC require a clean dentin surface void of CW or IRM residues for a sufficient seal. In
particular, IRM residues in the dentin tubules have been reported to affect the microtensile
bond strength of adhesives because IRM contains eugenol, which acts as a radical scavenger
and inhibits the polymerization process of methacrylates [45,46]. CF, on the other hand,
does not present any type of dentin adhesion and is thus comparatively easy to remove in
bulk. Even if the material is impacted in retentive cavities, the removal of CF only requires
one sectioning procedure along the center of the restoration.
It should be mentioned that the significance of our results is limited by the in vitro
nature of this study, which does not account for the complex environment in the human
oral cavity. Since the extracted teeth were not subjected to masticatory forces, our results
do not include potential failures due to loss of marginal integrity, which may severely
affect the tightness of the restorations. The integrity of the restorative materials may also
be influenced by patient-specific factors, such as bruxism, oral hygiene, or dietary habits
inducing recurrent pH drops in the oral cavity. Although temporary filling materials only
remain in the access cavity for a short time, the loss of tightness induced by such conditions
may affect the sterility of the pulp chamber and hence endodontic success. Therefore,
further microleakage studies simulating different oral conditions in addition to in vivo
studies are necessary to confirm our results.

5. Conclusions
Based on our in vitro results, CW/SDR and CF provided the best results in terms of
coronal tightness. There are implications that CF or CW/SDR (used with a self-adhesive
dentin bonding agent) should be favored when sealing access cavities of previously re-
stored teeth, especially when the coronal restoration contains methacrylate components.
Regarding double-layer techniques, there seemed to be no differences in outcome between
the CW/KM and IRM/KM specimens. From a methodological point of view, our study
demonstrates that the assessment of bacterial penetration with MRSA as a tracer combined
with the evaluation of glucose leakage provides reliable results for judging the sealing
ability of restorative materials.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jcm12144762/s1, Figure S1: Dimensions of the conical cylinder
used for standardized hollow test specimens; Figure S2: Sample agar plates showing the minimum
inhibitory concentrations of ampicillin and ciprofloxacin.
Author Contributions: Conceptualization, M.K. and L.M.; methodology, S.N.W., L.M., K.T.O. and
M.K.; validation, S.N.W., L.M., K.T.O. and M.K.; formal analysis, S.N.W. and L.M.; investigation,
S.N.W. and L.M.; resources, M.K.; data curation, L.M. and M.K.; writing—original draft preparation,
S.N.W.; writing—review and editing, L.M., K.T.O. and M.K.; visualization, S.N.W. and K.T.O.;
supervision, M.K.; project administration, M.K. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
J. Clin. Med. 2023, 12, 4762 12 of 13

Institutional Review Board Statement: Not applicable (ethics approval not needed, No. 23-0431 KB,
LMU Munich).
Informed Consent Statement: Not applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study. These data
can be found here: Würsching: Sabina Noreen und Moser, Luise und Obermeier, Katharina Theresa
und Kollmuß, Maximilian: Microleakage and economic efficiency of restorative materials used for
temporization of endodontic access cavities. Open Data LMU https://doi.org/10.5282/ubm/data.382,
accessed on 23 May 2023.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Nair, P.N.R. On the Causes of Persistent Apical Periodontitis: A Review. Int. Endod. J. 2006, 39, 249–281. [CrossRef] [PubMed]
2. Giovarruscio, M.; Sauro, S.; Makeeva, I.; Foschi, F. Strategies to Reduce the Risk of Reinfection and Cross-Contamination in
Endodontics. Clin. Dent. Rev. 2019, 3, 8. [CrossRef]
3. Ørstavik, D.; Qvist, V.; Stoltze, K. A Multivariate Analysis of the Outcome of Endodontic Treatment. Eur. J. Oral Sci. 2004, 112,
224–230. [CrossRef] [PubMed]
4. Ray, H.A.; Trope, M. Periapical Status of Endodontically Treated Teeth in Relation to the Technical Quality of the Root Filling and
the Coronal Restoration. Int. Endod. J. 1995, 28, 12–18. [CrossRef]
5. Abbott, P.V. Factors Associated with Continuing Pain in Endodontics. Aust. Dent. J. 1994, 39, 157–161. [CrossRef]
6. Naoum, H.J.; Chandler, N.P. Temporization for Endodontics. Int. Endod. J. 2002, 35, 964–978. [CrossRef]
7. Chen, P.; Chen, Z.; Teoh, Y.-Y.; Peters, O.A.; Peters, C.I. Orifice Barriers to Prevent Coronal Microleakage after Root Canal
Treatment: Systematic Review and Meta-Analysis. Aust. Dent. J. 2023, 68, 78–91. [CrossRef]
8. Chailertvanitkul, P.; Abbott, P.V.; Riley, T.V.; Sooksuntisakoonchai, N. Bacterial and Dye Penetration through Interim Restorations
Used during Endodontic Treatment of Molar Teeth. J. Endod. 2009, 35, 1017–1022. [CrossRef]
9. De Castro, P.H.D.F.; Pereira, J.V.; Sponchiado, E.C.J.; Marques, A.A.F.; Garcia, L.D.F.R. Evaluation of Marginal Leakage of Different
Temporary Restorative Materials in Endodontics. Contemp. Clin. Dent. 2013, 4, 472–475. [CrossRef]
10. Odabas, M.E.; Tulunoglu, O.; Ozalp, S.O.; Bodur, H. Microleakage of Different Temporary Filling Materials in Primary Teeth. J.
Clin. Pediatr. Dent. 2009, 34, 157–160. [CrossRef]
11. McDougal, R.A.; Delano, E.O.; Caplan, D.; Sigurdsson, A.; Trope, M. Success of an Alternative for Interim Management of
Irreversible Pulpitis. J. Am. Dent. Assoc. 2004, 135, 1707–1712. [CrossRef] [PubMed]
12. Barthel, C.R.; Strobach, A.; Briedigkeit, H.; Göbel, U.B.; Roulet, J.F. Leakage in Roots Coronally Sealed with Different Temporary
Fillings. J. Endod. 1999, 25, 731–734. [CrossRef] [PubMed]
13. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 30th ed.; CLSI Supplement M100; Clinical and Laboratory
Standards Institute: Wayne, PA, USA, 2020.
14. CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, 3rd ed.; CLSI
Supplement M45; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2015.
15. Torabinejad, M.; Rastegar, A.F.; Kettering, J.D.; Pitt Ford, T.R. Bacterial Leakage of Mineral Trioxide Aggregate as a Root-End
Filling Material. J. Endod. 1995, 21, 109–112. [CrossRef] [PubMed]
16. Wuersching, S.N.; Diegritz, C.; Hickel, R.; Huth, K.C.; Kollmuss, M. A Comprehensive In Vitro Comparison of the Biological and
Physicochemical Properties of Bioactive Root Canal Sealers. Clin. Oral Investig. 2022, 26, 6209–6222. [CrossRef] [PubMed]
17. Xu, Q.; Fan, M.; Fan, B.; Cheung, G.S.P.; Hu, H. A New Quantitative Method Using Glucose for Analysis of Endodontic Leakage.
Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2005, 99, 107–111. [CrossRef] [PubMed]
18. Kollmuss, M.; Preis, C.E.; Kist, S.; Hickel, R.; Huth, K.C. Differences in Physical Characteristics and Sealing Ability of Three
Tricalcium Silicate-Based Cements Used as Root-End-Filling Materials. Am. J. Dent. 2017, 30, 185–189.
19. van Rossum, G.; Drake, F.L. Python 3 Reference Manual; CreateSpace: Scotts Valley, CA, USA, 2019.
20. Mergoni, G.; Ganim, M.; Lodi, G.; Figini, L.; Gagliani, M.; Manfredi, M. Single versus Multiple Visits for Endodontic Treatment of
Permanent Teeth. Cochrane Database Syst. Rev. 2022, 12, CD005296. [CrossRef]
21. Schwendicke, F.; Göstemeyer, G. Single-Visit or Multiple-Visit Root Canal Treatment: Systematic Review, Meta-Analysis and Trial
Sequential Analysis. BMJ Open 2017, 7, e013115. [CrossRef]
22. Sjögren, U.; Figdor, D.; Persson, S.; Sundqvist, G. Influence of Infection at the Time of Root Filling on the Outcome of Endodontic
Treatment of Teeth with Apical Periodontitis. Int. Endod. J. 1997, 30, 297–306. [CrossRef]
23. Smith, E.A.; Marshall, J.G.; Selph, S.S.; Barker, D.R.; Sedgley, C.M. Nonsteroidal Anti-Inflammatory Drugs for Managing
Postoperative Endodontic Pain in Patients Who Present with Preoperative Pain: A Systematic Review and Meta-Analysis. J.
Endod. 2017, 43, 7–15. [CrossRef]
24. Siqueira, J.F.J.; Rôças, I.N. Clinical Implications and Microbiology of Bacterial Persistence after Treatment Procedures. J. Endod.
2008, 34, 1291–1301.e3. [CrossRef] [PubMed]
J. Clin. Med. 2023, 12, 4762 13 of 13

25. Sundqvist, G.; Figdor, D.; Persson, S.; Sjögren, U. Microbiologic Analysis of Teeth with Failed Endodontic Treatment and the
Outcome of Conservative Re-Treatment. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 1998, 85, 86–93. [CrossRef]
[PubMed]
26. Gaeta, C.; Marruganti, C.; Ali, I.A.A.; Fabbro, A.; Pinzauti, D.; Santoro, F.; Neelakantan, P.; Pozzi, G.; Grandini, S. The Presence
of Enterococcus Faecalis in Saliva as a Risk Factor for Endodontic Infection. Front. Cell. Infect. Microbiol. 2023, 13, 1061645.
[CrossRef] [PubMed]
27. Kampfer, J.; Göhring, T.N.; Attin, T.; Zehnder, M. Leakage of Food-Borne Enterococcus Faecalis through Temporary Fillings in a
Simulated Oral Environment. Int. Endod. J. 2007, 40, 471–477. [CrossRef]
28. Stuart, C.H.; Schwartz, S.A.; Beeson, T.J.; Owatz, C.B. Enterococcus Faecalis: Its Role in Root Canal Treatment Failure and Current
Concepts in Retreatment. J. Endod. 2006, 32, 93–98. [CrossRef]
29. Waltimo, T.; Trope, M.; Haapasalo, M.; Ørstavik, D. Clinical Efficacy of Treatment Procedures in Endodontic Infection Control
and One Year Follow-up of Periapical Healing. J. Endod. 2005, 31, 863–866. [CrossRef]
30. Veríssimo, D.M.; do Vale, M.S. Methodologies for Assessment of Apical and Coronal Leakage of Endodontic Filling Materials: A
Critical Review. J. Oral Sci. 2006, 48, 93–98. [CrossRef]
31. Taylor, M.J.; Lynch, E. Microleakage. J. Dent. 1992, 20, 3–10. [CrossRef]
32. Souza, E.M.; Pappen, F.G.; Shemesh, H.; Bonanato-Estrela, C.; Bonetti-Filho, I. Reliability of Assessing Dye Penetration along
Root Canal Fillings Using Methylene Blue. Aust. Endod. J. 2009, 35, 158–163. [CrossRef]
33. Friedman, S.; Shani, J.; Stabholz, A.; Kaplawi, J. Comparative Sealing Ability of Temporary Filling Materials Evaluated by Leakage
of Radiosodium. Int. Endod. J. 1986, 19, 187–193. [CrossRef]
34. Timpawat, S.; Amornchat, C.; Trisuwan, W.R. Bacterial Coronal Leakage after Obturation with Three Root Canal Sealers. J. Endod.
2001, 27, 36–39. [CrossRef] [PubMed]
35. Kist, S.; Kollmuss, M.; Jung, J.; Schubert, S.; Hickel, R.; Huth, K.C. Comparison of Ozone Gas and Sodium Hypochlo-
rite/Chlorhexidine Two-Visit Disinfection Protocols in Treating Apical Periodontitis: A Randomized Controlled Clinical Trial.
Clin. Oral Investig. 2017, 21, 995–1005. [CrossRef] [PubMed]
36. Shemesh, H.; Souza, E.M.; Wu, M.-K.; Wesselink, P.R. Glucose Reactivity with Filling Materials as a Limitation for Using the
Glucose Leakage Model. Int. Endod. J. 2008, 41, 869–872. [CrossRef]
37. Demarco, F.F.; Cenci, M.S.; Montagner, A.F.; de Lima, V.P.; Correa, M.B.; Moraes, R.R.; Opdam, N.J.M. Longevity of Composite
Restorations Is Definitely Not Only about Materials. Dent. Mater. 2023, 39, 1–12. [CrossRef] [PubMed]
38. Beach, C.W.; Calhoun, J.C.; Bramwell, J.D.; Hutter, J.W.; Miller, G.A. Clinical Evaluation of Bacterial Leakage of Endodontic
Temporary Filling Materials. J. Endod. 1996, 22, 459–462. [CrossRef] [PubMed]
39. Blaney, T.D.; Peters, D.D.; Setterstrom, J.; Bernier, W.E. Marginal Sealing Quality of IRM and Cavit as Assessed by Microbiol
Penetration. J. Endod. 1981, 7, 453–457. [CrossRef]
40. Ciftçi, A.; Vardarli, D.A.; Sönmez, I.S. Coronal Microleakage of Four Endodontic Temporary Restorative Materials: An In Vitro
Study. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2009, 108, e67–e70. [CrossRef]
41. Tulunoglu, O.; Uçtasli, M.B.; Ozdemir, S. Coronal Microleakage of Temporary Restorations in Previously Restored Teeth with
Amalgam and Composite. Oper. Dent. 2005, 30, 331–337.
42. Križnar, I.; Seme, K.; Fidler, A. Bacterial Microleakage of Temporary Filling Materials Used for Endodontic Access Cavity Sealing.
J. Dent. Sci. 2016, 11, 394–400. [CrossRef]
43. Adnan, S.; Khan, F.R. Comparison of Micro-Leakage around Temporary Restorative Materials Placed in Complex Endodontic
Access Cavities: An In-Vitro Study. J. Coll. Physicians Surg. Pak. 2016, 26, 182–186.
44. Widerman, F.H.; Eames, W.B.; Serene, T.P. The Physical and Biologic Properties of Cavit. J. Am. Dent. Assoc. 1971, 82, 378–382.
[CrossRef] [PubMed]
45. Wongsorachai, R.N.; Thanatvarakorn, O.; Prasansuttiporn, T.; Jittidecharaks, S.; Hosaka, K.; Foxton, R.M.; Nakajima, M. Effect of
Polymerization Accelerator on Bond Strength to Eugenol-Contaminated Dentin. J. Adhes. Dent. 2018, 20, 541–547. [CrossRef]
[PubMed]
46. Fujisawa, S.; Kadoma, Y. Effect of Phenolic Compounds on the Polymerization of Methyl Methacrylate. Dent. Mater. 1992, 8,
324–326. [CrossRef] [PubMed]

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