doi:10.1111/iej.
12046
The effect of medicaments used in endodontic
regeneration on root fracture and microhardness
of radicular dentine
G. H. Yassen1,2, M. M. Vail3, T. G. Chu1 & J. A. Platt1
1
Department of Restorative Dentistry, Division of Dental Biomaterials, Indiana University School of Dentistry, Indianapolis, IN,
USA; 2Department of Pediatric, Orthodontic and Preventive Dentistry, Mosul University School of Dentistry, Mosul, Iraq; and
3
Department of Endodontics, Indiana University School of Dentistry, Indianapolis, IN, USA
Abstract
Yassen GH, Vail MM, Chu TG, Platt JA. The effect of
medicaments used in endodontic regeneration on root
fracture and microhardness of radicular dentine. International
Endodontic Journal, 46, 688–695, 2013.
Aim To investigate the effect of medicaments used in
endodontic regeneration on root fracture resistance
and microhardness of radicular dentine.
Methodology The root canals of mandibular
premolars (n = 180) were instrumented and random-
ized into three treatment groups and an untreated
control group. Each treatment group received either
triple antibiotic paste (TAP), double antibiotic paste
(DAP) or calcium hydroxide paste [Ca(OH)2] intraca-
nal medicament. Teeth were kept in saline for 1 week,
1 month or 3 months. After each time-point, 15 teeth
were randomly selected from each group and two root
cylinders were obtained from each tooth. One cylinder
was subjected to a fracture resistance test, and the
other cylinder was used for a microhardness test.
Two-way ANOVA and Tukey’s pairwise comparisons
were used for statistical analysis.
Introduction
Endodontic regeneration has been introduced as a
treatment option for immature permanent teeth with
Correspondence: Ghaeth H. Yassen, Department of Restor-
ative Dentistry, Division of Dental Biomaterials, Indiana Uni-
versity School of Dentistry, 1121 W. Michigan St.,
Indianapolis, IN 46202, USA (Tel.: +1-317-437-2240; fax:
+1-317-278-7462; e-mail: gyassen@iupui.edu).
688 International Endodontic Journal, 46, 688–695, 2013
Results For the microhardness test, the two-way
interaction between group and time was significant
(P < 0.001). The intracanal application of TAP and
DAP caused significant and continuous decrease in
root dentine microhardness after one (P < 0.05)
and 3 months (P < 0.001), respectively. The three-
month intracanal application of Ca(OH)2 significantly
increased the microhardness of root dentine
(P < 0.05). The time factor had a significant effect on
fracture resistance (P < 0.001). The three intracanal
medicaments caused significant decreases in fracture
resistance ranging between 19% and 30% after
3-month application compared to 1-week application.
Conclusion In this laboratory study, the 3-month
application of triple antibiotic paste, double antibiotic
paste or calcium hydroxide paste medicaments signifi-
cantly reduced the root fracture resistance of
extracted teeth compared to a 1-week application.
Keywords: calcium hydroxide, double antibiotic
paste, fracture resistance, microhardness, regenera-
tion, triple antibiotic paste..
Received 1 October 2012; accepted 7 December 2012
necrotic pulps (Thibodeau 2009, Lenzi & Trope
2012). The technique has been suggested to reduce
the risk of fracture associated with traditional apexifi-
cation procedures by inducing a functional dentino-
pulpal complex that may lead to increased length and
width of fragile immature roots (Jung et al. 2008).
One of the essential elements for a successful
endodontic regeneration protocol is the creation of a
bacteria-free biological environment inside the root
canal space through the use of intracanal antibacterial
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

medicaments. The most widely used intracanal
medicament in endodontic regeneration is the triple
antibiotic paste (TAP) described by Hoshino et al.
(1996), which is a mixture of metronidazole, cipro-
floxacin and minocycline (Lovelace et al. 2011, Miller
et al. 2012). However, double antibiotic paste (DAP),
which is a combination of only metronidazole and
ciprofloxacin, has been used successfully in endodon-
tic regeneration (Iwaya et al. 2001) and was sug-
gested as a substitute for TAP to avoid the
discoloration effect of minocycline (Trope 2010). Fur-
thermore, calcium hydroxide [Ca(OH)2] has also been
used to disinfect the canal during endodontic regener-
ation (Chen et al. 2012).
Acids are commonly added to some antibiotics to
maintain chemical stability, control tonicity or to
ensure physiological compatibility. However, long-term
exposure of dental hard tissues to acidic antibiotics
might cause demineralization and negatively affect
their mechanical properties. Minocycline, a component
in the TAP, has been found to chelate calcium and
demineralize dental hard tissues (Minabe et al. 1994,
Maruyama et al. 2008). Furthermore, enamel exposure
to aqueous tetracycline solutions for one and 25 h
caused dramatic and continuous reduction in microh-
ardness (Bjorvatn & Olsen 1982). Similarly, highly
alkaline Ca(OH)2 medicaments have been suggested to
significantly decrease root resistance to fracture within
1 month (Sahebi et al. 2010).
No previous studies have tried to explore the effect
of various endodontic regeneration medicaments
on the mechanical properties of root dentine. There-
fore, the aim of this study was to investigate longitu-
dinally the effect of medicaments used in endodontic
regeneration on root resistance to fracture and mi-
crohardness of radicular dentine. It was hypothesized
that the three root canal medicaments used in the
endodontic regeneration technique have no signifi-
cant effect on radicular dentine microhardness and
root fracture resistance at all time-points.
Materials and methods
Tooth selection and endodontic preparation
Extracted human mandibular single-rooted premolars
(n = 180) were selected after obtaining local univer-
sity IRB approval (IRB number; 1108006606). The
teeth were stored in 0.1% thymol solution at 4 °C
and used within 6 months after extraction. The
inclusion criteria were absence of caries, root cracks,
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd
Yassen et al. Effect of Regeneration medicaments on root fracture
restorations and previous endodontic treatments. Fur-
thermore, only teeth with similar mesio-distal and
bucco-lingual root dimensions (8%) were included.
An endodontic access cavity was prepared in each
tooth using a round bur and a high-speed handpiece.
The working length was determined by visualizing
the tip of a size 15 K-file (Dentsply Maillefer, Ballai-
gues, Switzerland) extending beyond the apical fora-
men and subtracting 1 mm from that length of the
file. The root canals were mechanically prepared
using EndoSequence 0.06 taper rotary instruments
(Brasseler, Savannah, GA, USA) to a master apical
size 70 file. Along with instrumentation, 1 mL of
5.25% NaOCl was used as an intracanal irrigant
between uses of each succeeding file. Furthermore,
the canals were finally rinsed with sterile saline using
a 27-gauge needle and 5-mL syringe to remove any
dentine debris remaining in the canal after instru-
mentation. Root canals were then dried with sterile
paper points (Hygienic, Akron, Ohio, USA).
Medicament application
The teeth were then randomly assigned to three treat-
ments groups (TAP, DAP and Ca(OH)2 paste) and one
control group. For the control group (n = 45), no
medication was applied in the canal. For the first
treatment group (n = 45), TAP was prepared by mix-
ing USP-grade antibiotic powders compounded of
equal portions of metronidazole, ciprofloxacin and
minocycline (Champs Pharmacy, San Antonio, TX,
USA) with distilled water (powder/liquid ratio of
3 : 1). The paste was applied to the canal spaces with
a sterile lentulo spiral in a slow-speed handpiece
(Dentsply Caulk, Milford, DE, USA) and tamped in the
canal space to the level of the cemento-enamel junc-
tion using various sizes of sterile pluggers. For the
second treatment group (n = 45), DAP was prepared
by mixing USP-grade antibiotic powders compounded
of equal portions of metronidazole and ciprofloxacin
(Champs Pharmacy, San Antonio, TX, USA) with dis-
tilled water (powder/liquid ratio of 2.5 : 1).The paste
was applied to the canals as described previously. For
the third treatment group (n = 45), Ca(OH)2 paste
was prepared by mixing Ca(OH)2 powder (Dentonics,
Monroe, NC, USA) with distilled water (powder/liquid
ratio of 2 : 1). The paste was applied to the canals as
described previously. The root canals of the three
treatment groups were sealed apically with flowable
composite (TPH3, Dentsply Caulk). Furthermore, the
access openings of all teeth were sealed with a
International Endodontic Journal, 46, 688–695, 2013 689
 Effect of Regeneration medicaments on root fracture Yassen et al.

thickness of at least 4 mm of Cavit (3MESPE, St Paul, Fracture resistance testing

MN, USA). Teeth in the four groups were kept in
Each 5-mm root cylinder was tested for the resistance
normal saline at 37 °C for 1 week, 1 month or
to fracture using a universal testing machine (Sintech
3 months. The solution was changed every week. The
Renew 1123, MTS, Eden Prairie, MN, USA). The
three time-points were selected to represent the
application technique of a vertical loading force to
intracanal application time of medicaments suggested
fracture used in this study was modified from that
clinically, which usually ranges between 1 and
used in a previous study (Sedgley & Messer 1992). A
11 weeks (Ding et al. 2009, Thibodeau 2009, Lenzi &
slow-speed carbide bur was used under water coolant
Trope 2012).
to shape the root canal access at the coronal end of
each root cylinder to accept the loading fixture. The
Preparation of root specimens root cylinders were positioned vertically on the lower
fixed platform of the universal testing machine with
After each storage period, 15 randomly selected teeth
the coronal face upward using a double-sided adhe-
were taken from each group. Thus, there were one
sive tape. A cylindrical loading fixture with a spheri-
control subgroup and three treatment subgroups at
cal tip (r = 1.9 mm) attached to the upper crosshead
each time-point. Then, the teeth were decoronated at
was lowered until the spherical tip rested in the pre-
the level of 0.5 mm radicular to the facial cemento-
pared coronal root seat. Then, a vertical loading force
enamel junction with a low-speed diamond saw
was applied at a cross head speed of 0.5 mm per min-
(Buehler Ltd., Lake Bluff, IL, USA) under water
ute until the root cylinder fractured. Fracture was
cooling. Then, two root cylinders were horizontally
identified in the study when an instantaneous and
sectioned from each root using a water-cooled
sharp drop of more than 25% of the applied load was
diamond saw (Buehler Ltd). A cervical 5-mm root
observed (Teixeira et al. 2004). The load at fracture
cylinder was used for fracture resistance testing, and
was measured and expressed in Newtons.
a middle 3-mm root cylinder was used for microhard-
ness testing. The root cylinders from the treatment
groups were irrigated with distilled water to remove Scanning electron microscopy (SEM)
the medicaments.
One fractured root specimen was randomly selected
from each group after each time-point for SEM anal-
Microhardness testing ysis to observe the presence of medicament remnants
and/or any morphological changes in root canal den-
The three-millimetre root cylinders were mounted on
tine. Each selected root specimen was irrigated with
special rods, and the coronal sides of the specimens
5 mL of distilled water, sonicated in de-ionized water
were polished using a Struers Rotopol 31/Rotoforce 4
for 3 min and desiccated for 48 h. Then, specimens
polishing unit (Struers, Cleveland, PA, USA) with
were sputter coated for 3 min with gold/palladium
1,200-, 2,400- and 4,000-grit papers (Struers) and
using a sputter coater (Polaron, Agawam, MA, USA),
finally using a 1-lm diamond polishing suspension
and images were taken with a JEOL 6390LV
(Struers). As a final cleaning step, the polished speci-
scanning electron microscope (Peabody, MA, USA) in
mens were sonicated in de-ionized water for 3 min.
secondary electron imaging mode. All images were
Microhardness measurements were performed using
taken from the root canal surface area of the
a Knoop Microhardness Tester (Leco, LM247,
specimens.
St. Joseph, MI, USA) on the polished side of each root
cylinder at 500 lm and 1000 lm from the pulp–den-
tine interface. At each depth, three indentations were
Statistical analysis
made using a 50-g load oriented perpendicular to the
indentation surface for 15 s. The indentations were Normal distribution of the data was tested using the
carefully observed in an optical microscope with a Shapiro–Wilk test. The effects of type of medicaments
digital camera and image analysis software, allowing and duration of treatment on fracture resistance and
the precise measurement of their diagonals. The rep- microhardness measurements were examined using
resentative hardness value for each specimen at each two-way ANOVA followed by Tukey’s pairwise compari-
depth was obtained as the mean of the results for the sons. A 5% level of statistical significance was applied
three indentations. for the analyses.

690 International Endodontic Journal, 46, 688–695, 2013 © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Yassen et al. Effect of Regeneration medicaments on root fracture

had significantly lower microhardness than the

Results
Microhardness
The two-way interaction between group and time
was significant (P < 0.001) at both 500 and
1000 lm from pulp–dentine interfaces (Table 1).
Time had a significant effect on the TAP groups at
both depths (P < 0.001), with significantly lower
microhardness for the 3-month group than for the
1-week (P < 0.001) and 1-month groups (P = 0.02
at 500 lm and P = 0.01 at 1000 lm) and lower mi-
crohardness for the 1-month group than for the 1-
week group (P = 0.0007 at 500 lm and P = 0.014
at 1000 lm). Time had a significant effect on the
DAP groups at both depths (P < 0.001), with signifi-
cantly lower microhardness for the 3-month group
than for the 1-week (P < 0.001 at both depths) and
1-month groups (P = 0.04 at 500 lm and P < 0.001
at 1000 lm) and lower microhardness for the 1-
month group than for the 1-week group (P < 0.001
at both depths). Time had a significant effect on the
Ca(OH)2 groups (P = 0.005 at 500 lm and
P = 0.045 at 1000 lm), with significantly higher
microhardness for the 3-month group than for the
1-week group (P = 0.0012 at 500 lm and P = 0.038
at 1000 lm). Time did not have a significant effect
on the control groups (P > 0.05).
The groups’ comparisons at each time-point are
also shown in Table 1. No significant difference was
found after 1 week (P > 0.05) at both depths. How-
ever, after 1 month, the TAP group and the DAP
group had significantly lower microhardness than the
Ca(OH)2 and control groups (P < 0.001 at 500 lm
and P < 0.0005 at 1000 lm). After 3 months, the
TAP and DAP groups had significantly lower microh-
ardness than the Ca(OH)2 and control groups at both
depths (P < 0.001). Furthermore, the control group
Ca(OH)2 group (P = 0.0003 at 500 lm and
P = 0.015 at 1000 lm).
Fracture resistance
The time factor had a significant effect on root frac-
ture resistance (P < 0.001). Time had a significant
effect on the TAP and DAP groups (Table 2), with
significantly lower fracture resistance for the 3-month
group than for the 1-week group (P = 0.017 for TAP
and P = 0.008 for DAP). Time had a significant effect
on the Ca(OH)2 groups, with lower fracture resistance
for the 3-month group than for the 1-week
(P < 0.001) and 1-month (P = 0.007) groups. The
3-month percentage decrease in fracture resistance of
the TAP, DAP and Ca(OH)2 treatment groups com-
pared to 1-week groups was 19%, 21% and 30%,
respectively. No significant difference was found
between the control groups at all time-points
(P > 0.05). The overall effect of type of medicaments
used on fracture resistance did not reach a significant
difference (P = 0.055) (Table 2). No significant differ-
ence was found between all groups after 1 week or
1 month (P > 0.05). However, a significantly lower
facture resistance was found in the Ca(OH)2
(P = 0.007) and DAP (P = 0.042) groups compared
to the control group after 3 months.
SEM
Scanning electron microscopy (SEM) images taken at
15009 magnification showed that TAP- and DAP-
treated root canal dentine were depleted of the smear
layer with open dentine tubules at all time-points
(Fig. 1a,b). Furthermore, no TAP remnants were
observed at all time-points. However, few DAP rem-
nants were observed at all time-points. SEM images of

Table 1 Mean (SD) of Knoop microhardness for roots treated with endodontic regeneration medicaments and a control group
for 1 week, 1 month and 3 months at 500 lm and 1000 lm from the pulp–dentine interface

500 lm from pulp to dentine interface* 1000 lm from pulp to dentine interface*

Group Week (KH) Month (KH) 3 months (KH) Week (KH) Month (KH) 3 months (KH)

Calcium hydroxide paste 52 (5) Ab 54 (5) Aab 57 (4) Aa 58 (5) Ab 61 (5) Aab 62 (4) Aa
Untreated (Control) 53 (4) Aa 53 (4) Aa 51 (4) Ba 60 (4) Aa 61 (4) Aa 58 (4) Ba
Double antibiotic paste 53 (5) Aa 46 (4) Bb 43 (5) Cc 59 (5) Aa 55 (4) Bb 51 (5) Cc
Triple antibiotic paste 52 (4) Aa 47 (3) Bb 43 (6) Cc 59 (5) Aa 55 (4) Bb 51 (6) Cc

*At each distance, different upper case letter indicates significant difference between different groups within the same time-point
and at each distance, different lower case letter indicates significant difference between the three time-points within the same
group.

© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 46, 688–695, 2013 691
 Effect of Regeneration medicaments on root fracture Yassen et al.

Table 2 Mean (SD) of load at fracture for premolar roots treated with endodontic regeneration medicaments and untreated
control group for 1 week, 1 month and 3 months

Group Week (Newton)* Month (Newton)* 3 months (Newton)*

Untreated (control) 843 (128) Aa 814 (167) Aa 788 (138) Aa

Calcium hydroxide paste 867 (121) Aa 777 (222) Aa 607 (175) Bb
Double antibiotic paste 807 (172) Aa 740 (143) Aab 641 (131) Bb
Triple antibiotic paste 829 (130) Aa 806 (155) Aab 676 (113) Ab

*Different upper case letter indicates significant difference between different groups within the same time-point and different
lower case letter indicates significant difference between the three time-points within the same group.

(a) (b)

(c) (d)

Figure 1 Representative SEM images from root canal surface of roots treated with various endodontic regeneration medica-
ments and untreated control group. (a) Three-month triple antibiotic paste (TAP)-treated root canal. (b) Three-month double
antibiotic paste (DAP)-treated root canal. (c) Three-month Ca(OH)2-treated root canal. (d) Three-month untreated control root
canal.

Ca(OH)2-treated root canals showed firmly attached
Ca(OH)2 deposits at all time-points (Fig. 1c). The root
canal dentine in the control group was covered with
a smear layer at all time-points (Fig. 1d).
Discussion
The importance of exploring the effect of medicaments
used in endodontic regeneration on root fracture
should not be overlooked because the application time
of intracanal medicaments reported in some cases of
endodontic regeneration is relatively long, which may
reach up to 11 weeks (Thibodeau & Trope 2007,
Thibodeau 2009). Additionally, the increase in root
wall thickness was found to be limited to mid- and/or
apical root structures in the majority of reported
endodontic regeneration cases (Bose et al. 2009,
Hargreaves & Law 2010, Jeeruphan et al. 2012, Lenzi
& Trope 2012, Nosrat et al. 2012) rather than the
cervical part of the root, which is the area prone to
fracture in treated immature teeth with necrotic pulps
(Cvek 1992). Therefore, in cases where root thicken-
ing in the cervical area is not achieved through
regeneration, the potential of further weakening the

692 International Endodontic Journal, 46, 688–695, 2013 © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

root structure through long-term use of medicaments
should be avoided.
The significant decrease in root microhardness at
both depths after one- and 3-month treatment with
TAP or DAP could be explained by the strong
demineralization effect of these medicaments because
dentine hardness has been correlated with mineral
concentration (Kinney et al. 2003). A recent study
suggested that TAP and DAP caused significant
demineralization of superficial radicular dentine after
only 1-week application (Yassen et al. 2013). On the
other hand, Ca(OH)2 caused a gradual increase in
root microhardness with time, and this increase was
significant at both depths after 3 months. This might
be explained by the denaturation of the collagen
matrix caused by the low molecular weight and
highly alkaline pH of Ca(OH)2 as hypothesized in pre-
vious studies (Andreasen et al. 2002, Leiendecker
et al. 2012). A recent study found that Ca(OH)2
caused significant collagen degradation of superficial
radicular dentine after only 1-week application (Yas-
sen et al. 2013). It is well known that the collagen
component is responsible for toughness of the hard
tissues (Wang et al. 2001). Therefore, the compro-
mised collagen matrix in the more mineralized
dentine could lead to a more brittle and less tough,
even though harder, substrate. This could explain an
accelerated fatigue crack propagation during cyclic
stresses and an increase in the susceptibility of root
fracture in Ca(OH)2-treated root canals (Andreasen
et al. 2006). Increased susceptibility to fracture was
obvious in this study’s 3-month fracture resistance
data.
Three-month application of endodontic regenera-
tion medicaments caused a significant reduction in
fracture resistance of root specimens ranging
between 19% and 30% when compared to 1-week
application. The significant reduction in fracture
resistance after Ca(OH)2 application for 3 months
agrees with previous studies that found a significant
reduction in root fracture strength after 84 and
100 days of Ca(OH)2 application, respectively (An-
dreasen et al. 2006, Rosenberg et al. 2007). How-
ever, the reduction in fracture resistance after
1-month application of Ca(OH)2 did not reach a sig-
nificant difference in this study. A recent systematic
review of the literature found inconclusive data
regarding the effect of Ca(OH)2 exposure for 1
month or shorter on the mechanical properties of
radicular dentine (Yassen & Platt 2013). The signifi-
cant reduction in fracture resistance of root speci-
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd
Yassen et al. Effect of Regeneration medicaments on root fracture
mens after 3-month application of TAP and DAP
compared to 1-week application might be explained
by the demineralization effect of these acidic pastes
on radicular dentine, which was confirmed by the
microhardness data. The mineral component in den-
tine contributes to the strength of the tooth struc-
ture, and the relatively long-term exposure of
radicular dentine to antibiotic pastes might be the
reason for the significant reduction in root resis-
tance to fracture observed in this study. The effect
of intracanal medication on root fracture might be
more detrimental in vivo due to the very wide
immature root canals that may lead to a higher
amount of medicament per canal wall surface area
compared to this laboratory study. Furthermore, the
irrigation protocols suggested during endodontic
regeneration techniques might cause further reduc-
tion in root strength. EDTA, which is usually rec-
ommended during endodontic regeneration (Galler
et al. 2011, Miller et al. 2012), was found to signifi-
cantly reduce the radicular dentine microhardness
(De-Deus et al. 2006) and root resistance to fracture
(Uzunoglu et al. 2012).
The SEM images showed heavy Ca(OH)2 deposits
on Ca(OH)2-treated root canal dentine at all time-
points. On the other hand, TAP- and DAP-treated
dentine showed open tubules with no evidence of a
smear layer, which may further support the suggested
demineralization effect of these pastes. In addition to
the well-known antibacterial role of TAP and DAP,
the demineralization effect of these antibiotic pastes,
as suggested in this study, might play an additional
significant role in the creation of an environment
conducive to attachment of host stem cells on root
canal surfaces and exposure of collagen fibres and
various growth factors during endodontic regenera-
tion. A relatively recent retrospective study found that
endodontic regeneration cases treated with TAP had
significantly thicker root walls compared to regenera-
tion cases treated with Ca(OH)2 (Bose et al. 2009),
which might be explained by the root canal surface
conditioning effect of TAP observed in this study. If
Ca(OH)2 is to be used as a disinfectant during end-
odontic regeneration, the use of an intensive irriga-
tion protocol is recommended to remove Ca(OH)2
remnants and create a conditioned root canal surface.
On the other hand, the need for such an extensive
irrigation protocol should be reconsidered when TAP
or DAP is used for endodontic regeneration. However,
further in vivo studies are needed to substantiate these
suggestions.
International Endodontic Journal, 46, 688–695, 2013 693
 Effect of Regeneration medicaments on root fracture Yassen et al.
It is noteworthy to mention that endodontic regen-
eration cases usually require minimal or no instru-
mentation. However, root canal instrumentation was
performed in this study to standardize the internal
dimensions of roots before microhardness and fracture
resistance testing.
Conclusions
The null hypothesis stated that the three root canal
medicaments used in endodontic regeneration tech-
niques have no significant effect on radicular dentine
microhardness and root fracture resistance at all
time-points was rejected. The three-month application
of TAP, DAP and Ca(OH)2 medicaments significantly
reduced the in vitro root fracture resistance of man-
dibular premolars compared to a 1-week application.
Furthermore, TAP and DAP caused significant and
continuous decreases in microhardness of root den-
tine after one- and 3-month intracanal application.
Further studies are required to optimize the applica-
tion time of endodontic regeneration medicaments
and explore the advantage and disadvantages of the
demineralization effect of TAP and DAP suggested in
this study on endodontic regeneration techniques.
References
Andreasen JO, Farik B, Munksgaard C (2002) Long-term cal-
cium hydroxide as a root canal dressing may increase risk
of root fracture. Dental Traumatology 18, 134–7.
Andreasen JO, Munksgaard EC, Bakland LK (2006) Compari-
son of fracture resistance in root canals of immature sheep
teeth after filling with calcium hydroxide or MTA. Dental
Traumatology 22, 154–6.
Bjorvatn K, Olsen HC (1982) The effect of penicillin- and tet-
racycline-containing medicaments on the microhardness
of human dental enamel. An in vitro study. Acta Odontolog-
ica Scandinavica 40, 299–305.
Bose R, Nummikoski P, Hargreaves K (2009) A retrospec-
tive evaluation of radiographic outcomes in immature
teeth with necrotic root canal systems treated with
regenerative endodontic procedures. Journal of Endodontics
35, 1343–9.
Chen MY, Chen KL, Chen CA, Tayebaty F, Rosenberg PA,
Lin LM (2012) Responses of immature permanent teeth
with infected necrotic pulp tissue and apical periodontitis/
abscess to revascularization procedures. International End-
odontic Journal 45, 294–305.
Cvek M (1992) Prognosis of luxated non-vital maxillary inci-
sors treated with calcium hydroxide and filled with
gutta-percha. A retrospective clinical study. Endodontics
and Dental Traumatology 8, 45–55.
694 International Endodontic Journal, 46, 688–695, 2013
De-Deus G, Paciornik S, Mauricio MH (2006) Evaluation of
the effect of EDTA, EDTAC and citric acid on the microh-
ardness of root dentine. International Endodontic Journal
39, 401–7.
Ding RY, Cheung GS, Chen J, Yin XZ, Wang QQ, Zhang CF
(2009) Pulp revascularization of immature teeth with api-
cal periodontitis: a clinical study. Journal of Endodontics
35, 745–9.
Galler KM, D’Souza RN, Federlin M et al. (2011) Dentin con-
ditioning codetermines cell fate in regenerative endodon-
tics. Journal of Endodontics 37, 1536–41.
Hargreaves K, Law A (2010) Regenerative Endodontics. In:
Hargreaves K, Cohen S, eds. Cohen’s Pathways of the Pulp,
10th edn. Mosby: Elsevier, pp. 602–20.
Hoshino E, Kurihara-Ando N, Sato I et al. (1996) In-vitro
antibacterial susceptibility of bacteria taken from infected
root dentine to a mixture of ciprofloxacin, metronidazole
and minocycline. International Endodontic Journal 29,
125–30.
Iwaya SI, Ikawa M, Kubota M (2001) Revascularization of
an immature permanent tooth with apical periodontitis
and sinus tract. Dental Traumatology 17, 185–7.
Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L,
Khewsawai P, Hargreaves KM (2012) Mahidol study 1:
comparison of radiographic and survival outcomes of
immature teeth treated with either regenerative endodon-
tic or apexification methods: a retrospective study. Journal
of Endodontics 38, 1330–6.
Jung IY, Lee SJ, Hargreaves KM (2008) Biologically based
treatment of immature permanent teeth with pulpal
necrosis: a case series. Journal of Endodontics 34,
876–87.
Kinney JH, Marshall SJ, Marshall GW (2003) The mechani-
cal properties of human dentin: a critical review and
re-evaluation of the dental literature. Critical Reviews in
Oral Biology and Medicine 14, 13–29.
Leiendecker AP, Qi YP, Sawyer AN et al. (2012) Effects of
Calcium Silicate-based Materials on Collagen Matrix Integ-
rity of Mineralized Dentin. Journal of Endodontics 38, 829–
33.
Lenzi R, Trope M (2012) Revitalization procedures in two
traumatized incisors with different biological outcomes.
Journal of Endodontics 38, 411–4.
Lovelace TW, Henry MA, Hargreaves KM, Diogenes A
(2011) Evaluation of the delivery of mesenchymal stem
cells into the root canal space of necrotic immature teeth
after clinical regenerative endodontic procedure. Journal of
Endodontics 37, 133–8.
Maruyama H, Aoki A, Sasaki KM et al. (2008) The effect of
chemical and/or mechanical conditioning on the Er:YAG
laser-treated root cementum: analysis of surface morphol-
ogy and periodontal ligament fibroblast attachment. Lasers
in Surgery and Medicine 40, 211–22.
Miller EK, Lee JY, Tawil PZ, Teixeira FB, Vann WF Jr
(2012) Emerging therapies for the management of trau-
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd

matized immature permanent incisors. Pediatric Dentistry
34, 66–9.
Minabe M, Takeuchi K, Kumada H, Umemoto T (1994) The
effect of root conditioning with minocycline HCl in remov-
ing endotoxin from the roots of periodontally-involved
teeth. Journal of Periodontology 65, 387–92.
Nosrat A, Homayounfar N, Oloomi K (2012) Drawbacks and
unfavorable outcomes of regenerative endodontic
treatments of necrotic immature teeth: a literature review
and report of a case. Journal of Endodontics 38, 1428–34.
Rosenberg B, Murray PE, Namerow K (2007) The effect of
calcium hydroxide root filling on dentin fracture strength.
Dental Traumatology 23, 26–9.
Sahebi S, Moazami F, Abbott P (2010) The effects of short-
term calcium hydroxide application on the strength of
dentine. Dental Traumatology 26, 43–6.
Sedgley CM, Messer HH (1992) Are endodontically treated
teeth more brittle? Journal of Endodontics 18, 332–5.
Teixeira FB, Teixeira EC, Thompson JY, Trope M (2004)
Fracture resistance of roots endodontically treated with a
new resin filling material. Journal of the American Dental
Association 135, 646–52.
Thibodeau B (2009) Case report: pulp revascularization of a
necrotic, infected, immature, permanent tooth. Pediatric
Dentistry 31, 145–8.
© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd
Yassen et al. Effect of Regeneration medicaments on root fracture
Thibodeau B, Trope M (2007) Pulp revascularization of a
necrotic infected immature permanent tooth: case report
and review of the literature. Pediatric Dentistry 29, 47–
50.
Trope M (2010) Treatment of the immature tooth with a
non-vital pulp and apical periodontitis. Dental Clinics of
North America 54, 313–24.
Uzunoglu E, Aktemur S, Uyanik MO, Durmaz V, Nagas E
(2012) Effect of ethylenediaminetetraacetic Acid on root
fracture with respect to concentration at different time
exposures. Journal of Endodontics 38, 1110–3.
Wang X, Bank RA, TeKoppele JM, Agrawal CM (2001) The
role of collagen in determining bone mechanical proper-
ties. Journal of Orthopaedic Research 19, 1021–6.
Yassen GH, Platt JA (2013) The effect of nonsetting calcium
hydroxide on root fracture and mechanical properties of
radicular dentin: a systematic review of literature. Interna-
tional Endodontic Journal (In press).
Yassen GH, Chu TG, Eckert G, Platt JA (2013) The effect
of medicaments used in endodontic regeneration tech-
nique on the chemical structure of human immature
radicular dentin: an in vitro study. Journal of Endodontics
(In press).
International Endodontic Journal, 46, 688–695, 2013 695