Basic Research—Technology
Evaluation of Triple Antibiotic Paste Removal
by Different Irrigation Procedures
Julie A. Berkhoff, DDS, Paul B. Chen, BDS, Fabricio B. Teixeira, DDS, MS, PhD,
and Anibal Diogenes, DDS, MS, PhD
Abstract
Introduction: Regenerative endodontics aims to re-
establish a functional pulp-dentin complex. First, the
root canal system is disinfected primarily by irrigants
and medicaments. Triple antibiotic paste (TAP), a
commonly used intracanal medicament, has been shown
to be directly toxic to stem cells at concentrations greater
than 0.1 g/mL. Thus, its complete removal is a crucial step
in regenerative endodontic procedures. We hypothesized
that currently used irrigation techniques do not completely
remove TAP from root canal system. Methods: TAP was
radiolabeled by the incorporation of I125, and calcium hy-
droxide (Ultracal; Ultradent, South Jordan, UT) was radio-
labeled with Ca45. The intracanal medicaments were
placed into standardized human root segments and incu-
bated for 28 days at 37 C. Then, canals were irrigated
with EndoActivator (Dentsply, Tulsa, OK), passive ultra-
sonic irrigation, EndoVac (SybronEndo, Coppell, TX), or a
syringe/Max-i-Probe needle (Dentsply Rinn, Elgin, IL) using
a standardized irrigation protocol in a closed system.
Radioactivity levels (counts per minute values) were
measured for each tooth before and after the irrigation pro-
tocols. Furthermore, the canals were sequentially enlarged
and dentin samples collected and evaluated for radioac-
tivity. Data were analyzed with analysis of variance and
Bonferroni post hoc testing (P < .05). Results: Approxi-
mately 88% of the TAP was retained in the root canal sys-
tem regardless of the irrigation technique used (no
difference among groups). Furthermore, approximately
50% of the radiolabeled TAP was present circumferentially
up to 350 mm within the dentin. Conversely, up to 98% of
the radiolabeled intracanal calcium hydroxide was
removed, and most residual medicament was found pre-
sent in the initial 50 mm of dentin. Conclusions: Current
irrigation techniques do not effectively remove TAP from
root canal systems, possibly because of its penetration
and binding into dentin. However, calcium hydroxide is
effectively removed with significant less residual presence.
(J Endod 2014;40:1172–1177)
From the Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San
Antonio, TX 78229. E-mail address: Diogenes@uthscsa.edu
0099-2399/$ - see front matter
Copyright ª 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2013.12.027
1172 Berkhoff et al.
Key Words
Calcium hydroxide, Ca(OH)2, endodontics, irrigation, isotope, regenerative, TAP, triple
antibiotic paste
R egenerative endodontic treatment is a biologically based procedure aimed at re-
establishing a functional pulp-dentin complex. The key components include disin-
fection of the root canal and introduction of stem cells, growth factors, and scaffolds (1)
using treatment methods that impose minimal toxicity to stem cells (2). Immature teeth
are at risk for pulp necrosis because of trauma, dental anomalies, or caries. Historically,
root canal infections in these teeth have been treated with apexification procedures.
Although these procedures treat apical periodontitis, they do not favor the continued
root development often seen in teeth treated by regenerative endodontic procedures
(2, 3). Both apexification and regenerative endodontic procedures rely on adequate
disinfection of the root canal system for a successful outcome. It has been shown
that regenerative endodontic treatment is a stem cell–based therapy (4, 5). Thus, the
potential for tissue regeneration may well depend on stem cell survival and
differentiation capacity. From this perspective, the first critical step in a regenerative
procedure is to adequately disinfect the root canal system while creating a
microenvironment conducive to stem cell survival, proliferation, and differentiation.
Many disinfection protocols include the use of intracanal medicaments. Several
successful case reports used triple antibiotic (ciprofloxacin, metronidazole, and min-
ocycline) paste (TAP) in the root canal system for several weeks before the recruitment
of stem cells into the canal via induced bleeding (2). TAP is an effective antimicrobial
agent (6–8) that creates conditions suitable for tissue revascularization (9). However,
relatively little is known about the cellular effects of the paste at clinically used concen-
trations, and it is possible that different concentrations of TAP or irrigation protocols
may improve clinical outcomes. This latter point is very important because the TAP con-
stituents are administered directly into the root canal system, resulting in local concen-
trations many orders of magnitude greater than those found in systemic circulation after
oral administration. For example, circulating levels of ciprofloxacin (2 hours after a
500-mg capsule) peak at about 2.6 mg/mL in blood (10). However, concentrations
10,000 times greater (20 mg/mL ciprofloxacin in TAP) have been applied into the
root canal system in regenerative procedures (11). Thus, root canal concentrations
of antibiotics are about 5000- to 10,000-fold greater than circulating levels. This offers
the potential for nonselective toxicity of the antibiotics against host cells, with a partic-
ular concern regarding stem cells. A recent animal study found that TAP induced a mod-
erate inflammatory reaction in subcutaneous tissues (12). In addition, it has been
shown that the clinically used concentration of TAP is cytotoxic to the stem cells of
the apical papilla (13). These results provide a strong rationale for a study to determine
JOE — Volume 40, Number 8, August 2014

Figure 1. A schematic illustration of the fluid-closed system used for the aspiration and collection of the irrigants containing the radiolabeled medicaments. All
irrigants were delivered with a standardized rate of (2 mL/min) using a syringe pump and a 28-G Max-i-Probe needle placed within 1 mm from the apex, except for
the EV group, which had solutions delivered at the orifice and brought into the canal by the negative pressure created by its macrocannula placed within 1 mm from
the apex (not shown).
the optimal method for removing antibiotics from the root canal system
before the introduction of stem cells.
Calcium hydroxide (Ca[OH]2) is another commonly used medica-
ment in regenerative procedures, with several cases showing favorable
clinical outcomes (2). The therapeutic benefits of Ca(OH)2 have been
appreciated for decades in endodontics long before its use in regener-
ative procedures. Historically, Ca(OH)2 has been used as an antibacte-
rial medicament (14) and a pulp capping agent (15) and to promote
hard tissue formation in apexification procedures (16). Importantly,
it has been found recently to promote stem cell survival and prolifera-
tion (13). Thus, Ca(OH)2 represents another major class of intracanal
medicament used in regenerative procedures.
There are many contemporary irrigation modalities that can be
used to remove medicaments from the canal system. Sonic irrigation
with EndoActivator (EA) has unique appeal for regenerative treatment
because the tips are strong, flexible, and smooth, so they do not cut
dentin. It has been reported to effectively clean debris from the canals
and remove the smear layer even when used in curved canal systems
(17, 18). Passive ultrasonic irrigation (PUI) is a term that was first
described by Weller et al (19) in 1980. It is a technique that relies
on the ultrasonic activation of irrigants for the efficient removal of
debris and microorganisms (20). EndoVac (EV) is a negative-
pressure irrigation system that deposits fresh irrigant into the chamber
and aspirates it into the canal via an apically positioned microcannula.
This system has been shown to be more effective at removing debris,
particularly from the apical region, than conventional needle positive-
pressure irrigation (21–23). Positive-pressure syringe irrigation with
side-vented needles, such as Max-i-Probe (Dentsply Rinn, Elgin, IL),
is widely accepted and used by endodontists. It is inexpensive and allows
easy control of the depth of needle penetration and the volume of irri-
gant delivered (24). This method is proven most effective when the tip is
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Basic Research—Technology
in close proximity to the apex (25–27), a large volume of irrigant is
used (28), and the apical diameter is equal or larger than ISO 40
(29, 30). All of the aforementioned irrigation techniques are feasible
modalities to be used in regenerative endodontic treatment.
However, there is a gap in knowledge because the optimal irriga-
tion method for the removal of TAP and Ca(OH)2 from the root canal
system remains unknown. Therefore, the aim of the study was to eval-
uate the effectiveness of these irrigation techniques for the removal of
the 2 most commonly used intracanal medicaments, TAP and
Ca(OH)2, from simulated immature teeth with open apices.
Materials and Methods
Tooth Collection and Preparation
This study was approved by the Institutional Review Board of the
University of Texas Health Science Center at San Antonio, San Antonio,
TX. Extracted teeth were collected from the clinics of the University of
Texas Health Science Center at San Antonio School of Dentistry and
placed in 15 mmol/L sodium azide for 24 hours at 4 C followed by
copious irrigation with sterile saline and used for preparation of the
simulated immature roots with open apices.
A total of 36 roots from freshly extracted human teeth were pre-
pared for the study. The roots were sectioned from the crown at the level
of the orifice. The apical-most 3 mm of the roots was resected with a
#1557 bur (Komet, Rock Hill, SC) to remove ramifications and create
specimens with a centered canal and standardized length of 10 mm. The
canal lumen was initially cleared with a size 15 K-file and then enlarged
with ortho- and retrograde instrumentation using a ProTaper Finishing
File 1 (Dentsply, Tulsa, OK) to 8 mm. The walls were then paralleled and
the lumen standardized to 1.0 mm in diameter with a size 100 Light-
Speed LSX instrument (SybronEndo, Orange, CA).
Triple Antibiotic Paste Removal 1173
Basic Research—Technology
Figure 2. TAP is inefficiently removed from the canal of simulated immature
roots despite the use of different irrigation methods. Residual radiolabeled TAP
was measured after canals were irrigated with either positive pressure with a
side-vented needle (PP), positive pressure with sonic activation of the irrigants
using the EA, positive-pressure and negative-pressure irrigation using the EV,
or positive pressure with ultrasonic activation of irrigants (PUI). There was no
difference in labeled TAP removal among groups, with only approximately 20%
of the medicament being removed by the irrigation protocols. Data were
normalized to the initial total CPM values for each sample and analyzed with
1-way analysis of variance with the Bonferroni post hoc test and significance
set at P < .05 (n = 6 per group).
TAP Preparation and Radioactive Labeling
All procedures were performed under an experimental hood that
is approved for handling radioisotopes. A creamy mixture of TAP was
made by mixing equal amounts of the United States Pharmacopeia
grade form of each of the 3 antibiotics (ciprofloxacin, metronidazole,
and minocycline [1:1:1 ratio]) with sterile water (approximately 0.9
g antibiotic powder/mL of water). The antibiotic drugs were custom
labeled with a radioactive marker (I125) using the chloramine T method
previously described by our group (31) with approximately 1  105
counts per minute (CPM) per mL. The radioactivity of TAP was
measured in a Cobra II gamma counter (PerkinElmer Inc, Waltham,
MA), and the values were expressed in CPM. The use of gamma scintil-
lation counting permitted a nondestructive method for repeated
measuring of the radiolabeled TAP remaining in the root canal system.
Ca(OH)2 Radioactive Labeling
Ca(OH)2 paste (Ultracal; Ultradent, South Jordan, UT) was radio-
actively labeled by directly adding approximately 1  105/mL Ca45
isotope (PerkinElmer Inc). The Ca45-labeled Ultracal was measured
in a beta scintillation counter (Beckman Coulter, Indianapolis, IN).
The mixture was loaded into a new 1-mL syringe (BD, Oakville, Ontario)
fitted with a 28-G Max-i-Probe needle.
Medicament Placement
Immediately before labeled medicament placement, the apex was
closed with wax, and the root was irrigated with 1.5% NaOCl (20 mL)
followed by saline (20 mL). All specimens were irrigated with positive-
pressure irrigation using a Max-i-Probe, side-vented 28-G needle
placed within 1 mm of the apex. After irrigation, the canal was dried
with coarse paper points, and the specimens were divided into 2 groups
for medicament placement: TAP (n = 24) and Ca(OH)2 (n = 12). The
1174 Berkhoff et al.
Figure 3. Ca(OH)2 is efficiently removed from the canal of simulated imma-
ture roots. Residual radiolabeled Ca(OH)2 was measured after canals were
irrigated with either positive pressure with a side-vented needle (PP) or pos-
itive pressure with ultrasonic activation of irrigants (PUI). The ultrasonic acti-
vation of irrigants (PUI) resulted in a higher removal of Ca(OH)2 than positive
pressure alone. Both techniques resulted in the removal of greater than 90% of
the total labeled Ca(OH)2. Data were normalized to pretreatment CPM values
for each sample and analyzed with the Student t test with significance set at P <
.05 (n = 6 per group, *P < .05).
radiolabeled medicaments were placed into prepared canals (5 mL/
root segment), and the coronal aspect was sealed with wax. The spec-
imens were incubated for 28 days in a 37 C humidified incubator. In
addition, aliquots of the 2 labeled medicaments were directly placed
into sealed glass containers (n = 3/group) to serve as the control for
radioactivity decay over the study period.
Irrigation for Medicament Removal
After 28 days of incubation, the total CPM values were measured
for each specimen and the controls. This first measurement provided
a ‘‘pretreatment’’ sample before experimental removal of the radiola-
beled medicaments. Thus, each specimen served as its own pretreat-
ment control.
The TAP specimens were divided into 4 experimental groups
(n = 6 each) based on the irrigation technique:
1. Positive pressure using a syringe and a Max-i-Probe, side-vented
needle (PP group)
2. A Max-i-Probe side-vented needle followed by sonic activation with
the EA (EA group)
3. Negative pressure using the EV system (EV group)
4. A Max-i-Probe, side-vented needle with PUI (PUI group) using a file
adapter (SybronEndo), a size 15/.02 K-file (Dentsply), and a P5 ul-
trasonic unit (Acteon Satelec, Mount Laurel, NJ)
The Ca(OH)2 specimens were divided into 2 groups (n = 6 each):
1. The PP group
2. The PUI group
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 Basic Research—Technology

Figure 4. The distribution of radiolabeled intracanal medicaments within dentin. Dentin samples from different depths were collected by circumferential instru-
mentation. Residual radioactivity was measured to determine the percentage of labeled drug in each dentin depth sample fraction. Radiolabeled TAP was found
distributed throughout different depths of dentin equally in samples from the different irrigation methods. (A) Importantly, most of the radiolabeled TAP was found
in dentinal depths greater than 350 mm. Conversely, Ca(OH)2 was found significantly less within dentin than TAP (note the difference in the ordinate scales in A and
B). Furthermore, labeled Ca(OH)2 was found more superficially distributed with its greater concentration at 50 mm in dentin. (B) The use of PUI resulted in greater
removal of labeled Ca(OH)2. Data were normalized to the initial total pretreatment CPM values for each sample and analyzed with 2-way analysis of variance (depth
 irrigation) with the Bonferonni post hoc test and significance set at P < .05. Data are presented as the mean percentage of pretreatment CPM  standard de-
viation (n = 6 per group, *P < .05).

After the 28-day incubation period, the coronal seal was removed,
and the canals were irrigated with saline (20 mL) followed by 17%
EDTA (20 mL) and a final flush of saline (5 mL). All the irrigation pro-
tocols were standardized with the use of a computer-controlled syringe
pump (KD Scientific, Hayward, CA) programmed to deliver 2 mL of so-
lution per minute (Fig. 1). For the EA group, after every 5 mL of irriga-
tion, the irrigant was activated for 30 seconds with the tip positioned
within 1 mm from the apex. For the PUI group, after every 5 mL of irri-
gation, the irrigant was passively activated for 30 seconds at 50% power
without engaging the file (size 15 K-flex) against the dentinal walls. For
the EV group, the irrigants were delivered at the canal orifice through an
18-G needle and collected by aspiration using the EV macrocannula
positioned within 1 mm from the apex.
Throughout irrigation, the solutions were collected using a high-
volume microsuction tip attached to a closed fluid suction apparatus for
radioactivity containment (Fig. 1). The irrigant samples were collected
in a removable, disposable reservoir. Radioactivity measurements were
made for an aliquot of irrigant from each specimen. Between speci-
mens, the suction apparatus was flushed with saline to clear any residual
medicament from the suction tip and tubing.
Dentin Shavings Collection
To verify the amount of medicament remaining in dentin, the api-
cal wax was removed, and incremental instrumentation was performed
using Peezo reamers sizes 3, 4, 5, and 6 (Brasseler USA, Savannah, GA),
equivalent to ISO 110, 130, 150, 170, respectively. This instrumentation
allowed for stepwise removal of circumferential dentin to a depth of 50,
150, 250, and 350 mm, respectively. Radioactivity was measured from
the collected dentin shavings. In addition, total radioactivity was then
measured from the remaining specimen to determine whether radiola-
beled medicaments were still found in the root.
Data Analysis
Radioactivity measurements were normalized to the initial pre-
treatment CPM for each specimen. The total radioactivity recovered
in the irrigant solution is presented as the mean and standard deviation
of the percentage of pretreatment CPM. For dentin analysis, the data
(mean and standard deviation) are presented as a cumulative percent
of total radioactivity for each dentinal depth analyzed. Data for the total
remaining labeled medicament were analyzed with 1-way analysis of
variance with the Bonferroni post hoc test for multigroup comparison
for the TAP experiments and the Student t test for 2-group comparison
for the Ca(OH)2 experiments. Data for the percentage of the labeled
medicament in each dentinal depth were analyzed with 2-way analysis
of variance with the Bonferroni post hoc test. For all tests, significance
was set at P < .05 using Prism 6 (Graph Pad, La Jolla, CA).
Results
Radiolabeled TAP was difficult to remove from the root canal sys-
tem, and there was no statistical difference for removing labeled TAP
among the 4 irrigation groups (Fig. 2). Indeed, the mean percentage
removal of radiolabeled TAP was quite similar for the positive-
pressure (20.9%  9.9%), EA (14.4%  5.8%), EV (15.3% 
5.5%), and PUI (22%  13.2%) groups.
In contrast, the radiolabeled Ca(OH)2 was removed about 4-fold
more effectively than labeled TAP from the treated root canal systems
(Fig. 3). Moreover, significantly more Ca(OH)2 was removed with the
use of PUI technique (96.8%  2.5%) compared with the Max-i-
Probe irrigation needle (87.4%  13.6%).
Further analysis of the collected dentin shaving revealed that
greater than 85% of the labeled TAP remained in the dentin to a depth
greater than 350 mm with no significant difference among the irrigation
groups for samples collected at different dentinal depths (Fig. 4A).
Conversely, only approximately 5%–7.5% of the labeled Ca(OH)2 re-
mained in dentin with significantly greater removal of the labeled
Ca(OH)2 in the PUI group (Fig. 4B). A comparison of the irrigation re-
covery data (Fig. 3) versus dentin shavings data (Fig. 4) indicated that
the majority of the Ca(OH)2 in the root canal system was removed by the
irrigation step.
Discussion
TAP is a well-established antimicrobial agent shown to be highly
effective against endodontic pathogens (8, 9). It has been used in the
majority of regenerative endodontic procedures (2). However, it has

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Basic Research—Technology
been shown to have detrimental effects on the stem cells necessary for
regenerative treatment success (13). A recent study showed that this
medicament when used at currently used concentrations has an adverse
effect on stem cell survival even after attempts to remove them from the
root canal system (32). Although it has been successfully used in regen-
erative procedures, its toxicity may be one of the factors leading to poor
predictability of continued root development and re-establishment of
vitality responses in these cases (2). Moreover, TAP can directly affect
dentin including significant staining because of minocycline (33),
demineralization (possibly because of its very low pH of 3) (34),
and reduced microhardness and fracture resistance (35). Together,
these data indicate that TAP has both beneficial antimicrobial efficacy
and several potential adverse effects on the microenvironment of the
root canal system. Therefore, it is imperative that the TAP be adequately
removed from the canal space once it has served the antimicrobial pur-
pose.
In the current study, it was shown that TAP is not adequately
removed during regenerative procedures despite the use of different
irrigation techniques. A method was used in this study that covalently
linked radioactive iodine (tracer) as a way of tracking residual antibi-
otic medicament. This approach permitted the detection of residual
labeled drugs. Although this technique allows for greater sensitivity
and quantification, it does discriminate which of the 3 antibiotics in
the composition of the paste diffused into dentin. Nonetheless, more
than 80% of the antibiotic paste was localized within dentin despite at-
tempts to remove it with copious irrigation using different methods. This
very high diffusion and affinity for dentin may explain why this drug
combination leads to the minocycline-mediated staining often seen ex-
tending to the cementum layer. Indeed, most of the labeled TAP was
found in depths greater than 350 mm. Thus, the remaining effects of
this medicament on stem cell biology and the clinical outcome in regen-
erative endodontic procedures requires further investigation and war-
rants careful consideration by clinicians.
Ca(OH)2 is another well-established antimicrobial agent (36). Its
removal by different irrigation techniques has been extensively studied
using different methodologies (37). Thus, it was used as a control in
experiments to provide a comparative benchmark for the removal of
TAP. Unlike TAP, it was found to have no detrimental effects on stem
cells at clinically relevant concentrations and may even potentiate pro-
liferation either directly or after its removal (dentin conditioning) (13).
Nonetheless, it has been established that Ca(OH)2 can also unfavorably
alter radicular dentin, leading to decreased fracture resistance when
used for periods that far exceed its typical 1-month application in regen-
erative endodontic procedures (34, 38–40). Therefore, like TAP, it is
important to adequately remove Ca(OH)2 once its antimicrobial goal is
achieved. This study showed that Ca(OH)2 was almost entirely removed
with irrigation alone, and the remaining medication minimally
penetrated the dentin. For the Ca(OH)2 removal experiments, we
limited the testing to positive pressure (Max-i-Probe group) and PUI
(PUI group) because of the very high removal rates detected by both
techniques, resulting in approximately 90% and 95% removal,
respectively. Positive pressure is the most widely used irrigation
technique, and ultrasonic activation has been shown more effective
for removing Ca(OH)2 (37). The relatively small percentage that re-
mained was confined more superficially in dentin. It is possible that
this residual Ca(OH)2 in dentinal tubules underlies its proliferative ef-
fect on stem cells when cultured on dentin (41) or by solubilization of
dentin-derived growth factors (42, 43). Thus, there is greater than 4-
fold removal of Ca(OH)2 than TAP from simulated immature teeth
canal systems.
In conclusion, TAP appears to have high diffusion and retention
within dentin regardless of removal efforts with different irrigation
1176 Berkhoff et al.
methods. On the other hand, most of the labeled Ca(OH)2 was
adequately removed. Thus, remaining medicaments within dentin
have the potential to prolong their antibacterial effects but also increase
the likelihood of undesirable stem cell toxicity. The concentration and
formulation of these drugs must be optimized to provide maximum anti-
microbial effect while creating a microenvironment that fosters stem
cells proliferation and differentiation. The remaining effects of medica-
ments on stem cell biology, disinfection, and the clinical outcome in
regenerative endodontic procedures require further investigation and
warrants careful consideration by clinicians.
Acknowledgments
Supported by the Department of Endodontics at the University
of Texas Health Science Center at San Antonio.
The authors deny any conflicts of interest related to this study.
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