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Dental Traumatology 2015; 31: 374379; doi: 10.1111/edt.

12181

The effects of radicular dentine treated with


double antibiotic paste and
ethylenediaminetetraacetic acid on the
attachment and proliferation of dental pulp
stem cells

Ki Wan Kim1,*, Ghaeth H. Yassen2,*, Abstract Aim: This study explored the effects of dentine treated with
Ygal Ehrlich1, Kenneth Spolnik1, two concentrations of double antibiotic paste (DAP) and ethylenediamine-
Jeffrey A. Platt2, L. Jack Windsor3 tetraacetic acid (EDTA) on the attachment and proliferation of dental pulp
1
Department of Endodontics, Indiana University stem cells (DPSCs). Materials and Methods: Radicular dentine samples
School of Dentistry; 2Division of Dental were prepared with identical dimensions and randomized into six groups
Biomaterials, Department of Restorative (n = 4). Four groups were treated with double antibiotic paste (DAP) at
Dentistry, Indiana University School of concentrations of 500 mg ml 1 or 1 mg ml 1 with or without EDTA. The
Dentistry; 3Department of Oral Biology, Indiana other two groups were treated with EDTA only or received no treatment.
University School of Dentistry, Indianapolis, IN, DPSCs were seeded on each dentine sample (10 000 cells per sample). Lac-
USA
tate dehydrogenase activity assays were used to calculate the attached
DPSCs after 1 day of incubation. Water soluble tetrazolium assays were
performed to investigate DPSCs proliferation on the treated dentine sam-
Key words: Dental pulp stem cells; double ples after three additional days of incubation. Two-way ANOVA followed by
antibiotic paste; endodontic regeneration; TukeyKramer tests was used for statistical analyses (a = 0.05). Results:
ethylenediaminetetraacetic acid; radicular den-
tine
Dentine treated with 1 or 500 mg ml 1 of DAP followed by EDTA caused
significant increases in DPSCs attachment compared to the dentine treated
Correspondence to: L. Jack Windsor, with the DAP alone. The 500 mg ml 1 of DAP with or without EDTA
Department of Oral Biology, DS 271 Indiana caused significant reductions in DPSCs proliferation. However, the treat-
University School of Dentistry, 1121 W.
ment of dentine with 1 mg ml 1 of DAP did not have significant negative
Michigan St., Indianapolis, IN 46202, USA
Tel.: +1-317-274-1448 effects on DPSCs proliferation regardless of the use of EDTA.
Fax: +1-317-278-1411. Conclusion: The use of 1 mg ml 1 of DAP followed by 10 min of irriga-
e-mail: ljwindso@iu.edu tion with EDTA in endodontic regeneration procedure may have no nega-
Accepted 11 April, 2015 tive effects on the attachment and proliferation of DPSCs.
*These authors contributed equally to this
work.

Traumatic dental injuries and developmental dental (TAP) (7, 8), which is a mixture of ciprofloxacin, met-
anomalies are the main etiological factors of immature ronidazole and minocycline. However, the presence of
permanent teeth with pulpal necrosis (1). Regenerative minocycline in TAP has been associated with tooth dis-
endodontics (pulp revascularization) has been proposed coloration (8, 9). A double antibiotic paste devoid of
to induce root development and to improve various minocycline (ciprofloxacin/metronidazole) was found to
clinical outcomes compared to other traditional treat- have comparable antibacterial effects against endodon-
ments such as calcium hydroxide apexification and arti- tic pathogens (10) and has been successfully used in
ficial apical barrier technique using mineral trioxide endodontic regeneration (11). The second essential step
aggregate (2, 3). in endodontic regeneration therapy is to optimize the
The first essential step in endodontic regeneration biological environment inside the root canal for regen-
therapy is to disinfect the root canal with intensive eration by root canal irrigation with EDTA (12) and
chemical challenge through irrigation with sodium induction of bleeding to create natural scaffold and
hypochlorite and application of intracanal medicament provide enough cells inside the root canal (4, 13).
for 14 weeks (4). The most commonly used intracanal To optimize endodontic regeneration therapy, cells
medicaments during endodontic regeneration procedure inside the root canal must be able to attach and prolif-
are calcium hydroxide (5, 6) and triple antibiotic paste erate on dentine surfaces (14, 15) as well as differentiate

374 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Attachment of pulp stem cells to treated dentine 375

into odontoblast-like cells (16). Recent in vitro studies (Champs Pharmacy, San Antonio, TX, USA) with
found that the clinically used concentrations of various 1 ml of sterile water. This 500 mg ml 1 concentration
antibiotic medicaments such as TAP and DAP had of DAP was selected because it is the minimum
direct toxic effects on stem cells from apical papillae amount of DAP required to create a pasty consistence
(SCAP) (17) and pulp fibroblasts (18). Therefore, the that can be applied clinically into the root canal. The
use of lower concentrations of TAP and DAP (0.1 pulpal surface of the dentine slabs in the third and
1 mg ml 1) has been recommended to avoid the cyto- fourth groups was treated with 400 ll of a watery con-
toxic effects of these medicaments (4, 17). On the other sistency of DAP generated by mixing 50 mg of equal
hand, EDTA has been proposed to promote cell sur- portions metronidazole and ciprofloxacin with 50 ml of
vival during endodontic regeneration (12) due to its sterile water. This 1 mg ml 1 concentration of DAP
ability to demineralize superficial dentine (19), expose was selected because it is the concentration of DAP
dentine organic matrix (20), improve dental pulp stem suggested to be used during endodontic regeneration
cells (DPSCs) adhesion (21) and release dentine endog- according to a recent in vitro study (15). The pulpal
enous growth factors (22). No previous studies have surface of the dentine slabs in the fifth and sixth
explored the ability of EDTA to modify the attachment groups was exposed to 400 ll of sterile water. After
and proliferation of stem cells on dentine treated with that, all the dentine slabs were incubated for 1 week at
an intracanal antibiotic medicament. The aim of this 37C with an approximately 100% relative humidity.
study was to investigate the effects of dentine treated The 1-week dentine treatment with DAP was selected
with two concentrations of DAP with or without because it is the shortest intracanal application time of
EDTA irrigation on the attachment and proliferation medicaments recommended by the American Associa-
of DPSCs. tion of Endodontists (AAE) during endodontic regener-
ation procedure (23). After 1-week incubation, the
DAP-treated dentine slabs were rinsed with distilled
Materials and methods
water until there was no visible DAP. The pulpal sur-
face of dentine slabs from groups one, four, and six
Tooth selection and dentine sample preparation
was then slowly irrigated with 20 ml of 17% EDTA
Intact single-rooted human teeth were selected after (Henry Schein, Melville, NY, USA) for 10 min. The
obtaining Indiana University Institutional Review irrigation time of EDTA was selected based on a current
Board approval (IRB number: 1212010183). The teeth clinical regeneration protocol (4). Finally, each dentine
were stored at 4C in 0.1% thymol solution and used slab was rinsed with 500 ll of phosphate-buffered saline
within 6 months after extraction. Twenty-four dentine (PBS) and transferred into individual wells of a sterile
slabs (4 mm width 9 4 mm length 9 1 mm thickness) non-treated low attachment 96-well plate (Thermo Sci-
were obtained from the cervical half of each root using entific, Waltham, MA, USA) with the treated side of
a low-speed diamond saw (Isomet, Buehler, Lake Bluff, each dentine slab facing upward.
IL, USA) under running distilled water. The outer non-
pulpal surface of each specimen was flattened using an
DPSCs culture and seeding on dentine
automatic polishing unit (Struers, Cleveland, PA, USA)
with 500-grit paper (Struers). The pulpal side of each DPSCs (Cook General BioTechnology, Indianapolis,
dentine slab was polished using a polishing unit with IN, USA) were used in this study. The protocol used
1200-, 2400- and 4000-grit papers (Struers) and finally to harvest these cells has been previously described
using a diamond polishing spray (1 lm; Struers). Pol- (24). Furthermore, the ability of these cells to express
ished dentine slabs were sonicated for 3 min in neutral various mesenchymal cell surface markers and to differ-
detergent solution and rinsed for an additional 3 min entiate into various tissues in the appropriate differenti-
with deionized water. All the samples were examined ation medium has been established in previous reports
under light microscopy, and any dentine slab with (24, 25). DPSCs were cultured in low glucose (1 g l 1)
cracks was discarded and replaced. Finally, dentine Dulbeccos modified Eagles medium (DMEM) supple-
slabs were washed with sterile water, placed individually mented with 10% fetal bovine serum (FBS), 4 mM
in Whirl-pak bags (Sigma-Aldrich, St Louis, MO, L-glutamine, 2.5 g ml 1 fungizone, 100 unit ml 1 peni-
USA) containing a moist cotton pellet, gas sterilized cillin, and 50 g ml 1 gentamicin for 7 days at 37C,
with ethylene oxide, and stored at 4C until used. and 5% CO2. Subconfluent cells between passages 3
and 5 were utilized in this study.
DPSCs were seeded on the treated pulpal surface of
Dentine treatment procedure
each dentine slab (10 000 cells per sample). After 24 h
The dentine slabs were removed from 4C and washed attachment period, the media containing the unat-
with sterile water for 2 min. Each dentine slab was tached cells were collected and the cells were lysed uti-
then placed in a well of a 48-well plate (BD Falcon, lizing lactate dehydrogenase activity (LDH) assays and
San Jose, CA, USA) with the pulpal surface facing used to determine the amount of unattached and
upward. Dentine slabs were randomized intro six attached DPSCs. Each dentine sample was then washed
groups (n = 4 per group). The first and second experi- with 100 ll of serum plus DMEM, and the attached
mental groups were treated with 400 ll of a thin pasty DPSCs on each dentine slab received 200 ll of DMEM
consistency of DAP generated by mixing 500 mg of containing 10% FBS and incubated for an additional
equal portions metronidazole and ciprofloxacin 3 days. After a 3-day culture period, water soluble

2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
376 Kim et al.

tetrazolium salts (WST-1) assays were performed to The effects of the DAP concentration and final irriga-
determine DPSCs proliferation on the treated dentine tion with EDTA on the attachment and proliferation
samples. of DPSCs were examined using two-way ANOVA fol-
lowed by TukeyKramer pairwise comparisons
(a = 0.05). The high control groups (DPSCs with no
LDH assays
dentine specimens) in both experiments were excluded
LDH permeability assays were utilized to measure the from the statistical model and only used as reference
release of lactate dehydrogenase from unattached groups to calculate the percentages of attachment and
DPSCs after lysis in a 96-well plate following 24 h for proliferation.
attachment. A 100 ll mix of reconstituted LDH pre-
pared according to the manufacturers protocol (Roche
Results
Applied Science, Indianapolis, IN, USA) was added to
each sample and incubated for 30 min at 23C (room For DPSCs attachment experiments, the interaction
temperature). The absorbance was measured at 490 nm between DAP concentration and EDTA was significant
in a microplate reader (Titertek, Multiskan MCC, Flow (P = 0.0006). Figure 1 shows that dentine treated with
Laboratories, McLean, VA, USA). The culture media 1 or 500 mg ml 1 of DAP with no EDTA had signifi-
from four dentine specimens without cells were used as cant reductions in the attachment of DPSCs compared
low controls. Maximum LDH release from 10 000 cells to untreated dentine (P = 0.0006 and P = 0.001,
was generated by adding 10 ll of the lysis solution pro- respectively). However, no significant differences were
vided by the manufacturer for 30 min (high control). found in the attachment of DPSCs between dentine
The percentages of unattached DPSCs after various treated with 1 and 500 mg ml 1 of DAP without
dentine treatments were calculated relative to total EDTA (P > 0.05). Dentine treated with 1 mg ml 1 of
amount of cells (DPSCs with no dentine specimens) DAP with no EDTA had significant reductions in the
according to the following equation: Unattached attachment of DPSCs compared to dentine treated with
DPSCs (%) = (experimental absorbance value low 1 mg ml 1 of DAP followed by EDTA (P = 0.008).
control absorbance value)/(high control absorbance Additionally, dentine treatment with 500 mg ml 1 of
value low control absorbance value) 9100. Finally, DAP without EDTA caused significant reductions in
the percentage of attached cells after various treatments the attachment of DPSCs compared to dentine treated
was calculated by subtracting the percentage of unat- with 500 mg ml 1 of DAP followed by EDTA
tached cells from the percentage of total cells. The per- (P = 0.005). However, no significant difference was
centage of total cells (both attached and unattached) found in the attachment of DPSCs between untreated
was considered 100%. dentine and dentine treated with EDTA only
(P > 0.05). Furthermore, no significant differences were
found in the attachment of DPSCs between dentine
WST-1 assays

WST-1 proliferation assays were used to measure the


activity of mitochondrial dehydrogenase of the DPSCs
attached to treated dentine surfaces. After 3 days of
incubation, DPSCs on each dentine slab were washed
with 2 ml of serum-free media and incubated with
10 ll WST-1 (Roche Applied Science, Penzberg, Ger-
many) and 100 ll serum-free DMEM for 2 h in a
humidified atmosphere at 37C and 5% CO2. After
2 h, 100 ll of reaction mixture from each well (dentine
slab) was transferred to a new 96-well plate, and a
spectrophotometer (Titertek) was used to measure the
optical absorbance of each well at 450 nm. The culture
media from the four dentine specimens without cells
were used as a low control. The maximum WST-1 sig-
nal from 10 000 cells was also determined (high con-
trol). The percentage of DPSCs proliferation on treated
dentine samples in comparison with 10 000 cells was
calculated according to the following equation: Prolif-
eration of attached DPSCs (%) = (experimental absor-
bance value low control absorbance value)/(high Fig. 1. The percentages (mean + SE) of DPSCs attachment to
control absorbance value low control absorbance radicular dentine evaluated by LDH assays after various
dentine treatments with or without DAP and EDTA. Within
value) 9100.
each DAP treatment concentration, different upper-case
letters indicate statistically significant differences between
Statistical analyses dentine treated with or without EDTA. Within each EDTA
treatment condition, different lower-case letters indicate
All data were checked for normality using the Shapiro statistically significant differences between dentin treated with
Wilk test, and the normality assumption was satisfied. various concentration of DAP.

2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Attachment of pulp stem cells to treated dentine 377

treated with EDTA and that treated with 1 or quently proliferate and differentiate (14, 26). Previous
500 mg ml 1 of DAP followed by EDTA (all studies have explored the effects of various irrigation
P > 0.05). solutions on the attachment of DPSCs to radicular
For DPSCs proliferation experiments, the interaction dentine (14, 21, 26, 27). However, the present study is
between DAP concentration and EDTA was not signif- the first attempt to investigate the effects of dentine
icant (P = 0.93). Furthermore, the overall EDTA effect treated with intracanal medicament followed by EDTA
was not significant (P = 0.15). Figure 2 shows that the on the attachment and proliferation of DPSCs.
treatment of dentine with 500 mg ml 1 of DAP with This study showed that the treatments of 1 or
no EDTA caused significant reductions in the prolifera- 500 mg ml 1 DAP without EDTA caused significant
tion of DPSCs compared to dentine treated with reductions in DPSCs attachment to dentine. This could
1 mg ml 1 of DAP without EDTA and untreated den- be explained by residual cytotoxic effects of DAP or
tine (P < 0.0001). However, no significant difference the changes in dentine surface topography that may
was detected between dentin treated with 1 mg ml 1 of compromise the attachment of DPSCs. A previous
DAP without EDTA and untreated dentine (P > 0.05). study has also shown that DAP concentrations ranging
The treatment of dentine with 500 mg ml 1 of DAP from 1 to 100 mg ml 1 had detrimental direct cytotoxic
followed by EDTA caused significant reductions in the effects on SCAP (17) and pulp fibroblasts (18). Fur-
proliferation of DPSCs compared to dentine treated thermore, 1-week application of DAP (pH=3.4) was
with 1 mg ml 1 of DAP followed by EDTA suggested to cause significant demineralization of the
(P > 0.027) and dentine treated with EDTA only dentine surface (28). The current study showed that
(P = 0.002). However, no significant difference was dentine treated with 1 or 500 mg ml 1 of DAP fol-
detected between dentin treated with 1 mg ml 1 of lowed by 10-min irrigation with EDTA caused signifi-
DAP followed by EDTA and that treated with EDTA cant improvements in DPSCs attachment compared to
only (P > 0.05). Furthermore, no significant differences dentine treated with DAP without EDTA. This could
were detected between dentine groups treated with be attributed to the ability of EDTA to wash out the
DAP followed by EDTA compared to dentine groups residual DAP present in dentine and to expose various
treated with the same concentration of DAP with no dentine matrix components and growth factors such as
EDTA (P > 0.05). transforming growth factor-b, fibroblast growth factor
2, and bone morphogenetic protein 2 (22, 29).
In the current study, no significant differences were
Discussion
found in the attachment of DPSCs between untreated
To create new dentine during endodontic regeneration dentine and dentine irrigated with EDTA. This finding
therapy, DPSCs or other stem cells must be able to is consistent with a previous study that found no signif-
attach to the treated root canal dentine and subse- icant differences in the attachment of DPSCs to radicu-
lar dentine regardless of the use of EDTA (14).
However, other studies have found that irrigation of
dentine with EDTA caused significant improvement in
the attachment of DPSCs (21) and pulp fibroblasts
(27). It is noteworthy to mention that previous studies
(14, 21, 27) investigated the ability of EDTA to
improve DPSCs and pulp fibroblast attachment to
dentine used relatively short EDTA irrigation times
(1560 s) compared to the current study (10 min).
However, 10 min of irrigation with EDTA was selected
in this study based on a current clinical recommenda-
tion of endodontic regeneration treatment (4). The long
EDTA irrigation time may be helpful in washing out
the remaining intracanal medicaments and improve
DPSCs attachment within the context of the endodon-
tic regeneration clinical protocol. However, dentine
irrigation with EDTA for long periods without pervi-
ous application of intracanal medicament may not be
beneficial to the attachment of DPSCs.
This study showed that dentine treated with
500 mg ml 1 of DAP had negative effects on the pro-
Fig. 2. The percentages (mean + SE) of DPSCs proliferation liferation of DPSCs even after 10-min irrigation with
on radicular dentine evaluated by WST-1 assays after various EDTA. This generally agrees with a recent study that
dentine treatments with or without DAP and EDTA. Within suggested that dentine treatment with a pasty concen-
each DAP treatment concentration, different upper-case tration of DAP (1000 mg ml 1) had deleterious effects
letters indicate statistically significant differences between on SCAP seeded in a synthetic scaffold within the root
dentine treated with or without EDTA. Within each EDTA
treatment condition, different lower-case letters indicate
canal lumen (15). The presence of a continuous residual
statistically significant differences between dentin treated with cytotoxic effects from dentine treated with DAP even
various concentration of DAP. after irrigation with EDTA may negatively affect

2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
378 Kim et al.

DPSCs proliferation. A recent study suggested that followed by 10 min of irrigation with EDTA may be
radicular dentine treated with 1 mg ml 1 or recommended to avoid any negative effects on the
1000 mg ml 1 of DAP for 2 weeks exerted residual attachment and proliferation of DPSCs.
antibacterial effects up to 1 month after DAP removal
(30). On the other hand, the current study showed that
dentine treated with 1 mg ml 1 DAP significantly Conflict of interest
improved DPSCs proliferation compared to the use of The authors deny any conflicts of interest related to
500 mg ml 1 of DAP regardless of the use of EDTA. this study.
This agrees with a recent study that reported that den-
tine treated with 1 mg ml 1 of DAP had no cytotoxic
effects on SCAP seeded in a synthetic scaffold within References
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