Clinical Research
First Evidence for Regeneration of the
Periodontium to Mineral Trioxide Aggregate in
Human Teeth
Andreas Bartols, Dr, MA,* Eleni Roussa, Prof, Dr Winfried Walther, Prof, Dr*
orfer, Prof, Dr
and Christof E. D
Abstract
Introduction: The aim of this study was to elucidate
whether the use of mineral trioxide aggregate (MTA)
in endodontic therapy in human teeth leads to the
same regeneration of the apical tissues as observed in
animals. Methods: Four human teeth were identified
in a policlinic that had been treated endodontically
with MTA and had to be extracted for other reasons
than just endodontic failure. All teeth were processed
for histologic and one for immunohistochemical ana-
lyses to analyze the histologic response of the periapical
structure to the former treatment with MTA. Results: All
identified teeth showed clinical and radiographic signs
of healing at the time of extraction. In the histologic
evaluation, all teeth showed a layer of cementlike tis-
sues at least on the MTA surface. Further double immu-
nofluorescence analyses for collagen type I and type III
revealed protein expression and colocalization of the 2
proteins, implicating formation of periodontal ligament-
like tissue, presumably fibers. Conclusions: Histologic
healing of the human periodontium to MTA corresponds
to the healing pattern shown in animal studies. Cement-
like tissues were formed on the surface of MTA, which
proves regeneration of the periodontal ligament. (J En-
dod 2017;43:715722)
Key Words
Cementum, endodontic therapy, mineral trioxide aggre-
gate, periodontal ligament, regeneration
From the *Dental Academy for Continuing Professional Development, Karlsruhe, Germany; Clinic for Conservative Dentistry and Periodontology, School for Dental
Medicine, Christian-Albrechts-University, Kiel, Germany; and Departments of Molecular Embryology and Neuroanatomy, Institute of Anatomy and Cell Biology, Faculty
of Medicine, University of Freiburg, Germany.
Address requests for reprints to Dr Andreas Bartols, Karlsruhe, Baden-Wurttemberg, DE 76135. E-mail address: andreas_bartols@azfk.de
0099-2399/$ - see front matter
Copyright 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.12.027
JOE Volume 43, Number 5, May 2017
U ltimate healing of the
periodontium after
therapy of inflamed
Signicance
This article provides rst evidence for regeneration
of the periodontium to MTA in human teeth. The
periapical tissues of end-
histologic and immunohistologic evaluation shows
odontic origin is the
regeneration of cementlike tissues and periodontal
regeneration of a healthy
ligamentlike tissues in teeth treated with MTA.
periodontal ligament
Therefore MTA provides the most favorable mode
(PDL) with surrounding
of healing in clinical endodontic application toward
sound alveolar bone. Min-
a restitutio ad integrum of the periapical tissues.
eral trioxide aggregate
(MTA) seems to be a mate-
rial of choice for retrograde root-end fillings after endodontic surgery (13) but also
for orthograde endodontic treatments of teeth with open apices (4) because of its favor-
able properties concerning its sealing ability (5), biocompatibility (6), and histologic
healing of surrounding alveolar tissues (1, 2). A series of in vitro studies showed a
superior sealing ability of MTA compared with other retrograde filling materials,
such as intermediate restorative material (IRM), SuperEBA (Harry J Bosworth Co,
Skokie, IL), and amalgam (5, 7). Moreover, studies have shown that MTA is not
cytotoxic (8) and provides a favorable environment for PDL fibroblast adhesion as
well as for PDL fibroblasts and cementoblast growth (6, 9). Histologic animal
studies have revealed that MTA frequently initiates superior healing quality of the
resected dentin surfaces (1, 10, 11). Using materials like IRM or SuperEBA provides
the ability to heal toward the filling material, in best cases without signs of
inflammation in the PDL space, but without regeneration of the PDL (1, 10). Only
bioceramic root repair material shows comparable regenerative properties with MTA
regarding the PDL (2).
Although animal studies have shown the superior healing properties of MTA to-
ward the regeneration of the PDL, proof of the same favorable histologic response after
endodontic therapy performed with MTA stands out in humans. A systematic review of
the histologic responses of the periodontium to MTA in 2013 could only identify histo-
logic animal studies (12). To our knowledge, any histologic proof of the reaction of the
surrounding tissues after endodontic application of MTA in human teeth is lacking to
date. This is mainly because of massive ethical implications associated with obtaining
histologic samples of healed teeth in humans. Although teeth without signs of healing
will certainly not contribute to showing the healing potential of MTA toward PDL regen-
eration in humans, it is very rare that a clinically and radiographically healed tooth has
to be removed so that a histologic sample can be obtained.
Regeneration of the Periodontium to MTA 715
Clinical Research
Therefore, we have documented all cases of endodontic surgery

staining for collagen

performed with MTA in the outpatient clinic of the Dental Academy

sectioning and H&E

Undecalcified sawing/

Undecalcified sawing/

Undecalcified sawing/

immohistochemical
Type of histologic

toluidine staining

toluidine staining

toluidine staining

Decalcified paraffin
for Continuing Professional Development, Karlsruhe, Germany, and fol-

evaluation
lowed up these cases until the rare event that a tooth had to be removed

grinding with

grinding with

grinding with

type I and III

for other reasons than endodontic failure.
The purpose of this study was to elucidate whether the use of MTA
in human teeth leads to the same regeneration of the apical tissues as

and
observed in animals and to analyze the mode of regeneration, if any,
of the PDL.

histologic

with MTA
sections

surface
No. of
Methods

2
3
A total of 4 patients were identified, each having 1 tooth that was
formerly treated with MTA (Angelus MTA; Angelus Dental Products In-

roots healed)

Partially (apical
dustry S/A, Londrina, Brazil) and had to be extracted. Three teeth un-

Radio-graphic

root healed)
Partially (distal
(distobuccal

healing but
and palatal

resorption)
advancing
derwent root-end surgery, and 1 tooth was treated because of dental

healing
Complete Complete
trauma. In the last case, MTA was placed as an apical and lateral barrier

lateral
Partially
in a tooth with an open apex and signs of advanced lateral root resorp-
tion. All teeth showed clinical and/or radiographic healing of the peri-
apical tissues at the time of extraction. Tooth characteristics and the

Partially

Partially

Partially
reasons for extraction are summarized in Table 1.

healing
Clinical
All teeth were carefully luxated and removed to prevent damage of
the root tips with the adhering tissues as much as possible. For obvious
ethical reasons, it was not possible to obtain a resection of surrounding

treatment to
extraction in
bone tissue including the tooth. After extraction, the teeth were imme-

Age of patient Time from

months
diately fixed in 10% formalin. Subsequently, the teeth were processed

43

25

26

21
for histologic and immunohistochemical analysis.

Antibodies and Reagents/Chemicals

The mouse monoclonal antibody against collagen type I
treatment
at time of

in years
(ab90395) and the rabbit polyclonal antibody against collagen type 80

41

51

20
III (ab7778) were purchased from Abcam (Cambridge, UK). Donkey
antirabbit or antimouse immunoglobulin G and Alexa Fluor 488 (Dia-
nova, Hamburg, Germany) or Alexa Fluor 568 (Germany) were used as
secondary antibodies. Fluoromount-G was purchased from Biozol
Reason for extraction

(Eching, Germany).
periodontitis with

periodontitis with
clinical symptoms

clinical symptoms
mesiobuccal root
TABLE 1. Case Characteristics of Investigated Mineral Trioxide Aggregate (MTA) Treatments

Subgingival tooth

Persistent signs of
Persistent apical

Persistent apical

Histologic Analysis
mesial root

infection
Three teeth were embedded in acrylic resin (Technovit 4071; Her-
fracture

aeus Kulzer, Wehrheim, Germany). The teeth were then cut in sections
using a nondecalcifying sawing-grinding technique (13, 14) along their
coronoapical axis so that the area of interest containing the MTA was
exactly in the middle of the section plane. Sections of 100- to 150-
Orthograde placement
Persistent apical Root-end surgery with

Persistent apical Root-end surgery with

Persistent apical Root-end surgery with

mm thickness were ground with a grinding machine to sections of

of MTA as artificial
Type of treatment

7 mm with grain sizes up to P1200 Federation Europeenne des

retrograde MTA

retrograde MTA

retrograde MTA

apexification

Fabricants de Produits Abrasifs classification. The sections were then

application

application

application

stained with toluidine blue. If possible, more lateral sections were cut
and evaluated if they contained MTA in the section plane. The apical
region was photographed with a digital camera (CFW 1312M; Scion,
Frederick, MD) mounted to a microscope (Axiophot 2; Zeiss, Jena,
Germany).
Root resorption
periodontitis

periodontitis

periodontitis

All apical surfaces were quantified for newly formed cementlike

after dental
Reason for
treatment

tissue (CLT) and PDL-like tissue at the root tips with ImageJ of the
trauma

Fiji distribution (National Institutes of Health, Bethesda, MD) (15).

For quantitative analyses, the apical region of every histologic section
was photographed with an overlapping series of pictures at 20
magni-
fication. All pictures were photo stitched with Hugin (Version:
H&E, hematoxylin-eosin.
no. Tooth

2016.0.0.3b4e2790cb90 built by Jan Dubiec) for complete panoramas

13

16

46

21

of the apical surfaces of the roots. For metric analyses, ImageJ was set to
the correct micrometer per pixel relation at 20
magnification. The
Case

apical surfaces of the roots were measured as well as the surface of

1

MTA and the surface of CLT in micrometers. Then, the rates of coverage

716 Bartols Dr et al. JOE Volume 43, Number 5, May 2017


with CLT on the complete apical surface, dentin, and MTA were
calculated.
Single and Double Immunouorescence
One tooth was decalcified for 28 days in 20% EDTA and embedded
in paraffin as described earlier (14). This tooth was then cut with a
microtome into serial 4-mm sections. One section with the MTA opti-
mally centered was stained with hematoxylin-eosin (H&E) for conven-
tional histologic analysis. For immunofluorescence, sections were
deparaffinized, rehydrated with water, and washed with phosphate-
buffered saline (PBS). Slides were placed in 0.1 mol/L citrate buffer
(pH = 6.0) and heated in a microwave oven for 10 minutes at 600 W
for antigen retrieval. Sections were washed with PBS 3 times
for 5 minutes each and blocked with 10% normal goat serum/0.1 Triton
X-100/PBS (Sigma-Aldrich, St. Louis, MO) for 2 hours at room temper-
ature. Subsequently, sections were incubated with primary antibody
against collagen type III (rabbit polyclonal antibody at dilution 1:300
in blocking solution) or collagen type I (mouse monoclonal antibody
at dilution 1:100 in blocking solution) overnight at 4 C. The following
day slides were washed with PBS 3 times for 5 minutes each and incu-
bated with donkey antirabbit Alexa Fluor 568 or donkey antimouse
Alexa Fluor 488 as secondary antibodies for 1 hour at room tempera-
ture. Slides were washed with PBS (3 times for 5 min), rinsed with aqua
bidest, and mounted with Fluoromount-G containing 40 ,6-diamidino-
2-phenylindole dihydrochloride for nuclear staining. Slides were
viewed with a Leica SP6 confocal microscope (Leica, Mannheim,
Germany).
Results
Clinical and Radiographic Evaluation
All 4 teeth within this evaluation showed at least partial radio-
graphic signs of healing at the time of extraction. In case 1, a maxillary
right canine (tooth 13, World Dental Federation classification) was
originally treated with an apicoectomy with retrograde MTA filling
because of clinical and radiographic persistent signs of apical peri-
odontitis (Fig. 1A). After the treatment, the tooth was in service for
43 months without clinical symptoms. Then, the tooth fractured subgin-
givally and could not be restored so it was extracted. At the time of
extraction, the tooth did not show signs of apical periodontitis on the
x-ray (Fig. 1B). In case 2, a maxillary right first molar (tooth 16)
was originally treated with root-end surgery for persistent symptomatic
apical periodontitis (Fig. 2A). Twenty-five months after treatment, the
tooth showed clinical signs of inflammation. The x-ray (Fig. 2B) showed
an apical periodontitis of the mesiobuccal root, whereas the distobuccal
and palatal roots were radiographically healed. In case 3, a mandibular
right first molar (tooth 46) was also treated with an apicoectomy
because of persistent signs of apical periodontitis after initial root canal
treatment (Fig. 3A). Afterward, the tooth was in service for 26 months
and was then removed because of persistent clinical symptoms. The x-
ray (Fig. 3B) showed apical periodontitis at the mesial root, whereas the
distal root was healed. In case 4, a maxillary left first incisor was
endodontically treated after a dental luxation trauma. Because of a
wide apical foramen and lateral resorption, the complete root canal
filling was performed with MTA. After the initial treatment, the tooth
was in service for 21 months and then showed clinical signs of inflam-
mation. The x-rays (Fig. 4AC) showed advancing lateral resorption of
the root but nearly intact apical conditions. The tooth was removed
because of pain symptoms deriving from the inflammation tissue
because of the lateral resorptive tissue.
JOE Volume 43, Number 5, May 2017
Clinical Research
Histologic Evaluation
For histologic evaluation, we used only roots that showed signs of
radiographic healing (Table 1). Overall, we evaluated 9 histologic sec-
tions in 4 extracted teeth (cases 14, Figs. 14). Morphometric analysis
of the apical surfaces of the root-resected teeth with retrograde MTA fill-
ings (cases 13) showed nearly complete coverage with calcified CLT
(mean coverages: case 1, 100% [standard deviation (SD) = 0.0];
case 2, 94% [SD = 0.6]; and case 3, 97% [SD = 2.8], Figs. 13). In
case 4, the apical surface was covered with 34% (SD = 0.0) of CLT
(Fig. 4). Therefore, the overall mean coverage of CLT on the complete
apical surfaces was 81% (SD = 27.4). The mean coverage of CLT on the
dentin surfaces was 100% (SD = 0.0), 93% (SD = 0.8), 96% (SD =
4.4), and 0% (SD = 0.0) in cases 1 through 4, respectively. Surpris-
ingly, in all cases, all of the MTA surfaces were completely covered
with CLT (100% [SD = 0.0]).
In case 1, the complete apical resection surface in all 3 histologic
sections was completely covered with CLT (marked with CLT in
Fig. 1CE). This was also the case for the dentin (marked with D in
Fig. 1CE) and the MTA (marked with MTA in Fig. 1CE) surfaces.
In case 2, in the distobuccal and the palatal roots, there was 1
evaluable histologic section of each root available. In both sections,
there was not a complete coverage of the resection surface with CLT
(black arrows in Fig. 2C and D), and the layers of the calcified tissues
were not as thick as in case 1. Although small regions of the dentin were
not covered with CLT, all surfaces of MTA were completely covered.
In case 3, 2 histologic sections of the distal root were evaluated. In
1 of the sections, the complete surface was covered with at least a thin
layer of CLT on the dentin and the MTA (Fig. 3D). In the other section
(Fig. 3C), a region on the dentinal surface was covered with a grain of
MTA where no CLT was seen (orange arrow in Fig. 3C). In both sec-
tions, the MTA was completely covered with CLT.
In case 4 (Fig. 4), the sections stained with H&E showed signs of
resorption over the dentinal surfaces (Fig. 4D). Only on the apically
placed MTA barrier was a layer of CLT observed. Figure 4D also shows
invasion of inflammatory tissue into the MTA identified by the dark blue
labeling of the cell nuclei. In case 4, we wanted to elucidate the molec-
ular composition of the CLT barrier on the MTA surface. As shown in
Figure 4E, immunofluorescence with antibodies against collagen I
(green) revealed that the tissue covering the MTA surface was strongly
immunopositive for collagen I and exhibited similar labeling intensity to
the native cementum, suggesting the presence of cellular cementum. In
some areas, both MTA and the CLT barrier were invaded by inflamma-
tion tissue.
In addition, double immunofluorescence using specific antibodies
against collagen type I (green, Fig. 4F and G) and collagen type III (red,
Fig. 4F and G) revealed labeling for both proteins at the periapical
area in connection with the CLT barrier on the MTA surface (arrows),
showing protein expression and colocalization (orange, overlay
Fig. 4F and G) of the 2 proteins and implicating the formation of
PDL-like tissue, presumably fibers.
Discussion
The present study is the first evidence for a mode of regeneration
of periapical tissue by means of formation of CLT in teeth treated with
MTA. This corresponds to histologic animal studies and therefore shows
for the first time the transferability of the animal model to humans.
Moreover, it was possible to histologically show the protein expression
of collagen type I and III at the surface of CLT, strongly suggesting signs
of regeneration of the PDL adjacent to MTA.
Regeneration of the Periodontium to MTA 717
Clinical Research

Figure 1. Case 1. (A) Preoperative radiograph of a maxillary right canine with symptomatic apical periodontitis. (B) Radiographic evaluation of a maxillary right
canine 43 months after apicoectomy and retrograde MTA filling revealed no signs of apical periodontitis. Acrylic resin sections from the tooth were labeled with
toluidine blue, and the histologic pictures of the black boxed area are shown in CE. (CE) Sections from different depths of section planes. Resected dentin (D)
and cementum surfaces as well as the MTA were covered by a continuous and homogenous layer of newly formed hard tissue structurally resembling dental
cementum (CLT). Scale bar: 50 mm. The table within the figure shows the different morphometric values of the corresponding histologic sections in CE.

Because the presence of bacteria in the root canal leads to the
inflammation of periapical tissues (16), a goal of surgical and nonsur-
gical endodontic treatment is to prevent leakage of bacteria and their
by-products to the surrounding apical tissues. The superior sealing abil-
ities of MTA has been convincingly shown in in vitro studies (5, 7).
Histologic animal studies have further demonstrated that MTA
apparently provides the properties for superior healing of the tissues
in contact to the material (13, 11) and that the histologic healing
correlates to radiologic signs of healing (2, 3). Clinical studies on
surgical as well as nonsurgical endodontic treatments confirm the
histologic findings in the sense of high rates on radiologic success
after the application of MTA (4, 17, 18). We shared this observation;
in our sample of teeth, all radiologically healed roots also showed
histologically restored apical tissues (ie, newly formed CLT and signs
of a newly formed PDL) in contact with MTA. Notably, the area of MTA
was always found to be covered with a layer of tissue (Figs. 1CE, 2C
and D, 3C and D, and 4DG) that clearly showed similar staining and
morphologic properties as root cementum (ie, CLT).
Several in vitro studies showed that MTA is not cytotoxic
(19, 20) compared with amalgam, IRM, SuperEBA, and Diaket
(3M ESPE, Neuss, Germany). Moreover, human periodontal
fibroblasts showed the densest growth (9) and normal function

718 Bartols Dr et al. JOE Volume 43, Number 5, May 2017

 Clinical Research

Figure 2. Case 2. (A) Preoperative radiograph of a maxillary right first molar with symptomatic apical periodontitis at all roots. (B) Radiographic evaluation of a
maxillary right first molar 25 months after root-end surgery and retrograde filling with MTA; the mesiobuccal root showed clear signs of inflammation, whereas the
periapical area of the (b) distobuccal and (c) palatal root were without pathological findings. Acrylic resin sections from the tooth were labeled with toluidine blue,
and the histology pictures of the black boxes b and c areas are shown in C and D, respectively. The resection area containing MTA and native dentin (D) surfaces
was covered by a continuous and homogenous layer of newly formed hard tissue, structurally resembling dental cementum (CLT). Only small areas were not
covered with CLT (black arrows). Scale bar: 50 mm. The table within the figure shows the different morphometric values of the corresponding histologic sections
in C and D.

JOE Volume 43, Number 5, May 2017 Regeneration of the Periodontium to MTA 719
Clinical Research

Figure 3. Case 3. (A) Preoperative radiograph of a mandibular right first molar with symptomatic apical periodontitis at both roots. (B) Radiographic evaluation
26 months after apicoectomy and retrograde MTA filling; apical periodontitis was clearly visible in the mesial root, whereas the distal root showed no signs of
inflammation. Acrylic resin sections from the distal root were labeled with toluidine blue, and the histology pictures of the black boxed area are illustrated at
different depths of the section plane in C and D. A layer of CLT was detected in contact to MTA, dentin (D), and dental cementum surfaces. In some areas,
the newly formed tissue contained lacunae with nonmineralized tissue (orange and black arrows). Scale bar: 50 mm. The table within the figure containing
the different morphometric values of the corresponding histologic sections in C and D.

720 Bartols Dr et al. JOE Volume 43, Number 5, May 2017

 Clinical Research

Figure 4. Case 4. Radiographic evaluation of a maxillary left first incisive after treatment of a luxation injury. (A) An intraoperative radiograph with apical peri-
odontitis. (B) The initial radiographic control immediately after placement of MTA and (C) 21 months later. Progressive lateral root resorption but a nonsympto-
matic periapical area was detected. Note the lack of MTA from the lateral resorption lacunae in C. The tooth was decalcified in EDTA and embedded in paraffin. The
black boxed area is shown in D in a section labeled with hematoxylin-eosin (D) for conventional histology. The apical root tip exhibits signs of resorption. An apical
barrier of calcified CLT was formed covering the MTA and the dentin (D) surfaces. IT, inflammation tissue invading the MTA. (EG) Double immunofluorescence
for collagen type I (green) and collagen type III (red) followed by confocal microscopy. Nuclear staining with 40 ,6-diamidino-2-phenylindole dihydrochloride. (E)
High magnification of the black boxed area in D. Collagen type Irich apical tissue in contact with MTA and dentin (D) and outlined with tissue expressing both
collagen type I (green) and collagen type III (red). (F and G) Higher magnification of the white boxed areas in E illustrating expression of (F and G) collagen type I
and (F0 and G0 ) collagen type III. (F00 and G00 ) The overlays show colocalization (orange) of the proteins. Arrows point to areas expressing both collagen types.
Scale bar: 50 mm and 150 mm in D. The table within the figure shows the different morphometric values of the corresponding histologic sections in D.

(20) when cultured with MTA in contrast to amalgam, Dyract
(Dentsply Detrey GmBH, Konstanz, Germany), IRM, and Super-
EBA. Periodontal fibroblasts also show attachment to MTA that
cannot be demonstrated with gutta-percha (21). Therefore,
MTA is not just an inert material but appears to be a material
with biological activity toward cementogenic regeneration and
PDL fibroblast proliferation (22). MTA enhances the probability
of regeneration of periapical tissues toward restitutio ad integrum
in endodontic treatment. In our histologically investigated cases,
we found such healing with signs of restored periodontal tissues.
To the best of our knowledge, there are no studies that attempted to
molecularly decipher the mode of regeneration of the PDL after the appli-
cation of MTA. It is described in several histologic animal studies that
structural similarity to the PDL can be observed in H&E staining between
newly formed CLT and periapical bone structures (2, 3, 10), but there
was no immunohistochemical proof for PDLs. Type I collagen is the
predominant collagen of cementum, comprising up to 90% of the
organic components of cellular cementum, and our
immunofluorescence results (Fig. 4F and G) show expression of collagen
I in the CLT covering the MTA area. Because the predominant collagens in
the PDL are type I and III (23, 24), single and double
immunofluorescence methods were used together with high-resolution
confocal microscopy to detect the expression of both collagen types.
With this method, we found regions of expression of both collagen types
I and III along the surface of the newly formed CLT pointing in the direc-
tion of the surrounding tissues. Because the surrounding bone could not
be harvested for histologic analyses and the PDL was disrupted during
extraction, these tissues remain incomplete, but the location and detec-
tion of collagen proteins strongly indicate a newly formed PDL on the
newly formed CLT adjacent to the MTA surface.
Even though it was possible to demonstrate signs of regeneration
of human periapical tissues for the first time using our histologic and
immunofluorescence techniques, we are aware of the limitations of
our study. Normally, there is no reason to remove clinically and radio-
graphically healed teeth after endodontic application of MTA, making
the extraction of such a tooth a very rare case. Therefore, there are
only a very limited number of cases. Moreover, for obvious ethical rea-
sons, it is not possible to remove the surrounding bone of apically
healed teeth for complete histologic analyses. The histologic analyses
will remain incomplete regarding the connection of possible regener-
ated PDL fibers to the bone.
The histologic healing of human periapical tissues after endodon-
tic placement of MTA corresponds to the healing pattern demonstrated
in animal studies. Also, in humans, CLT will be formed on the surface of
apically placed MTA as in animals. Moreover, it was possible to prove
the regeneration of the PDL on the surface of newly formed CLT.
Acknowledgments
The authors thank Mrs Marquardt for her excellent technical
assistance with the preparation of the histologic sections and Mrs
Feuerstein for her excellent technical assistance with the immuno-
histochemical processing.
The authors deny any conflicts of interest related to this study.
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JOE Volume 43, Number 5, May 2017