doi:10.1111/iej.
12738
Effect of MTA particle size on periapical healing
M. Torabinejad1, S. M. Moazzami2, H. Moaddel1, J. Hawkins3, C. Gustefson4, H. Faras5, K.
Wright6 & S. Shabahang1
1
Department of Endodontics, Loma Linda University, Loma Linda, CA, USA; 2Department of Endodontics, Mashad University,
Mashad, Iran; 3Private Practice, Kennewick, WA; 4Private Practice, Chico, CA, USA; 5Department of Endodontics, Kuwait
University, Kuwait City, Kuwait; and 6Department of Anatomy, Loma Linda University, Loma Linda, CA, USA
Abstract
Torabinejad M, Moazzami SM, Moaddel H,
Hawkins J, Gustefson C, Faras H, Wright K,
Shabahang S. Effect of MTA particle size on periapical
healing. International Endodontic Journal, 50, e3–e8, 2017.
Aim To examine the effect of reduction in MTA par-
ticle size on dento-alveolar and osseous healing in
dogs.
Methodology Root canals of 24 mandibular pre-
molars in four 2-year-old beagles were prepared and
filled with gutta-percha and sealer. Two to four weeks
later, during periapical surgery, the root-end cavity
preparations in these teeth were filled with either grey
ProRoot MTA or modified (reduced particle sizes with
faster setting time) MTA. The animals were sacrificed
Introduction
Excisional wound healing following periapical surgery
comprises osseous and dentoalveolar healing (Harrison
& Johnson 1997). Harrison & Jurosky (1992) created
osseous wounds in the mandibles and maxillas of rhesus
monkeys and evaluated alveolar healing histologically.
They found that alveolar healing progressed from the
endosteal surfaces towards the external surfaces of the
bony wound, resulting in formation of woven bone at
14 days and a functioning periosteum at 28 days.
Under ideal circumstances, dento-alveolar healing
results in complete regeneration of periradicular tissues
surrounding the roots (Harrison & Johnson 1997).
Correspondence: Shahrokh Shabahang, Department of End-
odontics, Loma Linda University, 11092 Anderson Street,
Prince Hall 4401, Loma Linda, CA 92350, USA (Tel.: (909)
558-4681, fax: (909) 558-0122, e-mail: shahrokh@shaba
hang.net).
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
4 months later. Degrees of inflammation, type of
inflammatory cells, fibrous connective tissue adjacent
to the root-end filling materials, cementum formation
over the resected roots and root-end filling materials
and bone healing were examined. Data were analysed
using the McNemar test.
Results No significant differences in healing of peri-
apical tissues were found when comparing ProRoot
MTA to a modified MTA containing reduced particle
sizes.
Conclusion Reducing the particle sizes of MTA did
not impact its biological properties.
Keywords: fast-set, MTA, periapical healing, root-
end filling material.
Received 12 October 2015; accepted 8 December 2016
The mechanism of dento-alveolar healing is not
fully understood. It is thought that undifferentiated
mesenchymal cells, fibroblasts and fibroblast-like cells
arise from the periodontal ligament and bone and
surround the root-end to begin the healing process.
The undifferentiated cells transform into mature
fibroblasts, cementoblasts and osteoblasts and begin
to reform the periradicular dento-alveolar apparatus
(Andreasen 1973). Cementum is laid down from the
outside edges of the root towards the centre of the
resected root-end. With most root-end filling materi-
als, cementum formation is limited to the resected
root-end and does not extend over the filling material
(Harrison & Johnson 1997).
Mineral trioxide aggregate (MTA) has many unique
characteristics that are consistent with an ideal material
for various endodontic applications, including its use as
root-end filling during apical surgery. The dento-alveo-
lar healing adjacent to MTA root-end fillings is unique
in that it results in regeneration of the periapical tissues,
International Endodontic Journal, 50, e3–e8, 2017 e3
 MTA particle size/PA healing Torabinejad et al.
comprising complete apical cementogenesis over MTA
(Torabinejad et al. 1997). The characteristics of apical
cementogenesis adjacent to MTA have been elucidated
in animal studies using cats (Maguire et al. 1998), dogs
(Torabinejad et al. 1995a,b) and monkeys (Torabinejad
et al. 1997). A potential limitation of MTA is its long set-
ting time (Torabinejad et al. 1995a,b). Several investi-
gators have modified MTA chemically to reduce its
setting time with variable success rates (Kogan et al.
2006, Ber et al. 2007, Huang et al. 2008). Addition of
new chemicals to MTA may impact the intrinsic charac-
teristics of the material. Therefore, new formulations of
MTA must be tested extensively before they can safely
be used in a clinical setting (Parirokh & Torabinejad
2010a,b).
Particle sizes of cements can affect their setting
time. A number of studies have evaluated the effect of
reducing the particle size of MTA or adding nanopar-
ticles to MTA and other cements (Saghiri et al. 2012,
2015, Akbari et al. 2013). These investigations have
shown that a reduction in setting times occurs by
either reducing the particle size or by incorporating
nanoparticles to these materials. Recently, a fast-set
MTA formulation (US Patent No. 8960576 B2) has
been developed by using high shear impingement of
MTA particles mixed with 100% ethanol under high
pressure and vacuum (Microfluidics, Newton, MA,
USA). The starting median particle size of ProRoot
MTA is 3.848 lm. After processing, the median parti-
cle size of ProRoot MTA is reduced approximately 10
times to 0.631 lm (US Patent No. 8960576 B2). The
new fast-set MTA is composed of 25% reduced parti-
cle size MTA and 75% of ProRoot MTA (Dental/
Dentsply International, Tulsa, OK, USA).
Currently, it is not known whether changing the set-
ting time of MTA by reducing its particle sizes can affect
its biological properties when used as a root-end filling
material. The aim of this study was to examine the effect
of reducing particle size of MTA on dento-alveolar and
osseous healing in dogs. The null hypothesis was that
changing the particle size of MTA does not impact
dento-alveolar and osseous healing.
Materials and methods
This animal experiment was performed in accordance
with protocols approved by the Loma Linda University
Animal Research Committee and the radiation safety.
Twenty-four roots of mandibular third and fourth pre-
molars of four 2-year-old beagle dogs were used.
Using a split-mouth design, 16 roots received fast-set
e4 International Endodontic Journal, 50, e3–e8, 2017
MTA and eight roots received ProRoot MTA as the
root-end filling material. Anaesthesia was obtained by
an initial intramuscular injection of 10 mg kg 1 of
tiletamine HCl and zolezapam HCl (Telazol, Fort
Dodge Animal Health, Overland Park, KS, USA) and
0.04 mg kg 1 atropine sulphate (Abbot Labs, Abbot
Park, IL, USA). The animals were then intubated and
maintained on inhalation anaesthesia with 1–3%
isoflurane (Forane, Ohmeda, Liberty Corner, NJ, USA)
and 1–2 L min 1 of oxygen for the remainder of the
procedure. Local anaesthesia was provided with a
buccal infiltration of 1.8 mL 2% lidocaine (Xylocaine,
Astra Pharmaceuticals, Wilmington, DE, USA) with
1:50 000 epinephrine.
After gaining occlusal access to the pulp chambers
of the experimental teeth, the pulps were extirpated
and the root canals were cleaned and shaped using
Flex-o-Files (Dentsply Tulsa Dental, Tulsa, OK, USA).
The canals were filled with warm vertical compaction
of gutta-percha and AH root canal sealer (Dentsply
Tulsa Dental). The coronal access cavities in all teeth
were then restored with amalgam (Ivoclar Vivadent,
Amherst, NY, USA). Two weeks after completion of
the root canal procedures, the dogs underwent the
first periapical surgery procedure on either the
mandibular right or left quadrant. After obtaining
anaesthesia, a full thickness mucoperiosteal buccal
flap with two releasing incisions (mesial of the third
and distal of the fourth premolars) was reflected. The
cortical bone covering the root-ends was removed
using a number 6 round bur in a high-speed hand-
piece using copious saline irrigation. The root-ends
were resected with a fissure bur in a high-speed hand-
piece with saline irrigation approximately 3 mm from
the apex at an angle approximately 60° to the long
axis of the root. Root-end cavities were then prepared
to a depth of 3 mm with a Vista P5 ultrasonic unit
(Vista Dental, Racine, WI, USA) and an S12/90 ultra-
sonic tip (Satelec, A-dec, Inc., Newberg, OR, USA).
Sixteen root-end cavities were filled with modified
MTA (containing reduced particle size) and eight were
filled with ProRoot MTA. The mucoperiosteal flaps
were repositioned and sutured with 4-0 silk sutures.
All animals were administered 0.01 mg kg 1
buprenorphine (Buprenex, Reckitt and Coleman Phar-
maceuticals, Richmond, VA, USA) subcutaneously for
pain control and 300 000 units of penicillin (Bicillin
C-R, Wyeth-Ayerst Laboratories, Philadelphia, PA,
USA) to prevent infection. After surgery, the animals
were placed on a soft diet. Two weeks after comple-
tion of the first periapical surgery, the second surgery
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
 Torabinejad et al. MTA particle size/PA healing

was performed on the contralateral premolars follow- Table 1 Histological findings in periradicular tissues of root-
ing similar protocols described for the first periapical ends filled with modified MTA or ProRoot MTA
surgery. The sutures were removed 2 weeks after Sig
each surgery. Case Type Modified Regular (P-Value)
The animals were euthanized 16 weeks after the
Degree of inflammation
second surgical procedure using an overdose of barbi- None 11/15 5/7 NS
turate (Euthasol, Western Medical Supplies, Arcadia, Mild 4/15 2/7 NS
CA, USA). After perfusion with 10% buffered forma- Moderate 0/15 0/7 NS
lin, mandibular block sections containing the premo- Severe 0/15 0/7 NS
Predominant cell
lar teeth and surrounding tissues were obtained.
Macrophage: M 0/15 0/7 NS
Specimens were demineralized in 5% formic acid and Polymorph: P 0/15 0/7 NS
then dehydrated in ascending concentrations of alco- Lymphocyte: L 4/15 2/7 NS
hol. After embedding in paraffin, 10–15 bucco-lingual Complete healing 11/15 5/7 NS
sections of 6 lm thickness were cut through the cen- Fibrous capsule formation 1/15 1/7 NS
Cementum over root-end
tre of the apical foramen along the long axis of each
None 0/15 1/7 NS
tooth. All sections were stained with haematoxylin Noncontinuous 4/15 1/7 NS
and eosin (H&E) and evaluated under a light Continuous 11/15 5/7 NS
microscope. Cementum over material
Degrees of inflammation, types of inflammatory None 3/15 1/7 NS
Noncontinuous 5/15 1/7 NS
cells, fibrous connective tissue adjacent to the root-
Continuous 7/15 5/7 NS
end filling materials, cementum formation over the New bone formation
resected roots and root-end filling materials, as well None 0/15 0/7 NS
as osseous healing, were evaluated according to modi- Partial 0/15 0/7 NS
fied criteria used in a previous investigation (Tora- Complete 15/15 7/7 NS

binejad et al. 1997). Three observers jointly examined

the specimens without knowledge of the designation
of the groups. Due to the split-mouth design in this
study, paired analyses were performed using the
McNemar test. Differences in proportions of histologi-
cal outcomes between MTA and modified MTA split-
mouth sites were compared with the McNemar test.
Results
A total of 15 modified MTA and seven ProRoot MTA
specimens were available for histological evaluation.
One sample of modified MTA and one sample of Pro-
Root MTA were lost during histological preparation of
the specimens. The histological findings of the avail-
able samples are summarized in Table 1. Eleven of 15
samples of modified MTA had no inflammation, whilst
five of seven samples of ProRoot MTA had no inflam-
mation. When inflammatory cells were present, lym-
phocytes were the predominant cells in all of the
examined samples. No significant differences were
observed for any of the histological outcomes when
comparing the paired regular MTA and fast-set MTA
sites (P = 1.0). Eleven of 15 samples of modified MTA
had complete healing (Fig. 1), normal periapical tis-
sues and no inflammation, whilst five of seven sam-
ples of ProRoot MTA had complete healing (Fig. 2). A
fibrous capsule was observed adjacent to one of the
modified MTA samples and in one of the ProRoot
MTA specimens. For all histological observations of
healing (e.g. fibrous capsule formation, cementum
over the root-end or new bone formation), no statis-
tics were computed because regular MTA and fast-set
MTA results were constants. Continuous cementum
formation was noted over resected roots in 11 of 15
samples of modified MTA and five of seven samples of
ProRoot MTA. Noncontinuous cementum formation
was observed over the dentinal surface of four of 15
samples of modified MTA and one of seven samples of
ProRoot MTA. One sample of ProRoot MTA had no
cementum formation over its resected root surfaces. A
continuous layer of cementum was found over the
root-end filling material in seven of 15 samples of
modified MTA and five of seven samples of ProRoot
MTA. Five of 15 samples of modified MTA had a non-
continuous layer of cementum formation over the
root-end filling material, whilst one of seven samples
of ProRoot MTA had a noncontinuous layer of
cementum over the root-end filling material. Three of
15 samples of modified MTA had no cementum for-
mation over the root-end filling material, whilst one
of seven samples of ProRoot MTA had no cementum

© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 50, e3–e8, 2017 e5
 MTA particle size/PA healing Torabinejad et al.
(a)
(b)
Figure 1 (a) A modified MTA specimen showing complete
dento-alveolar healing 16 weeks after periapical surgery.
Image at 4.09 magnification. (b) Continuous cementum (C)
formation over both resected root surfaces and MTA. Image
at 8.09 magnification.
formation over the root-end filling material. Complete
bone formation occurred after periapical surgery
regardless of the type of root-end filling material
placed in the root-end cavity preparations.
Discussion
The suitability of root-end filling materials is tested by
assessing their ability to prevent leakage, by assessing
their biocompatibility and by conducting usage tests
in animal and clinical studies. Numerous studies have
shown that MTA prevents leakage of dye, bacteria
e6 International Endodontic Journal, 50, e3–e8, 2017
(a)
(b)
Figure 2 (a) A ProRoot MTA specimen showing complete
dento-alveolar healing 16 weeks after periapical surgery.
Image at 4.09 magnification. (b) A continuous layer of
cementum (C) is observed over ProRoot MTA. Image at 109
magnification.
and endotoxin (Parirokh & Torabinejad 2010a,b). As
root-end filling materials are placed in intimate con-
tact with periradicular tissues, it is essential that a
potential root-end filling material be nontoxic and bio-
compatible. Several studies have shown that MTA is
a biocompatible material (Torabinejad & Parirokh
2010). Animal usage tests comparing MTA and other
commonly used root-end filling materials have shown
presence of less inflammation and better healing with
MTA compared to other root-end filling materials
(Glickman & Hartwell 2008). When MTA is used as a
root-end filling material, histological evaluation
reveals regeneration of periradicular tissues and for-
mation of new cementum over the MTA (Torabinejad
et al. 1995a,b, 1997, Maguire et al. 1998). Clinical
studies have demonstrated significantly better out-
comes when MTA is used as a root-end filling mate-
rial compared to other commonly used root-end filling
materials (Saunders 2008, Chong et al. 2009, von
Arx et al. 2010, 2014, Tsesis et al. 2013).
Despite favourable reports in experimental animals
and clinical trials, one of the disadvantages of Pro-
Root MTA is its long setting time. Reducing the set-
ting time of MTA makes it easier to handle the
material during placement, prevents its washout dur-
ing application and eliminates the need for a second
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd

visit to place a final restoration. The setting time of
MTA can be reduced either chemically or physically.
Reducing the setting time of MTA chemically has the
potential to change its intrinsic characteristics
(Saghiri et al. 2012). The method used to reduce the
particle size of MTA in this study utilizes high shear
impingement of MTA particles mixed with 100%
ethanol under high pressure and vacuum (Microflu-
idics). The starting median particle size of ProRoot
MTA is 3.848 lm. After processing, the median parti-
cle size of MTA is reduced approximately 10 times to
0.631 lm. This process results in an MTA formula-
tion that sets in less than a minute. This change in
setting is consistent with the findings of Saghiri et al.
(2012) who reduced the setting time of white MTA
by creating nanoparticles of this material. The results
are also consistent with the results of Akbari et al.
(2013) who reduced the setting time of MTA by add-
ing nanosilicates. Addition of small particles to
cements as fillers increases the surface areas of the
particles allowing the materials to more readily react
with liquids such as water, thereby reducing their set-
ting time (Akbari et al. 2013).
Reduction in particle sizes of MTA has the potential
to change its intrinsic characteristics and the new for-
mulation should be tested in in vitro and in vivo con-
ditions to evaluate this potential change (Akbari et al.
2013). An initial dye leakage study showed no signifi-
cant difference in linear penetration of the dye
between modified MTA and ProRoot MTA (Challenger
et al. 2015). An in vitro cell culture study showed
lack of cytotoxicity of modified MTA (preliminary
unpublished data). The focus of this experiment was
to histologically examine the effect of changing parti-
cle sizes of MTA on dento-alveolar and osseous heal-
ing in dogs. No significant differences in healing of
periapical tissues were found when comparing Pro-
Root MTA to an MTA with modified particle sizes.
Examination of the specimens for dento-alveolar heal-
ing revealed no significant differences between the
two materials with respect to degrees of inflammation,
types of inflammatory cells, fibrous connective tissue
adjacent to the root-end filling materials, cementum
formation over the resected roots or root-end filling
materials, or osseous healing when fast-set grey MTA
was compared to ProRoot MTA. These results are
generally consistent with those of previous investiga-
tions (Torabinejad et al. 1995a,b, 1997, Maguire
et al. 1998). One hundred per cent successful healing
was not achieved by either material in this or previ-
ous studies. There may be several variables
© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd
Torabinejad et al. MTA particle size/PA healing
attributing to this finding including operator variabil-
ity from case to case, anatomical variations (e.g. posi-
tion of the roots in the alveolus), variation in healing
amongst individual subjects and other factors.
Conclusion
Based on these results, it appears that changing the
particle sizes of MTA using high shear impingement
does not improve or negatively impact its biological
properties when used as a root-end filling material
during periapical surgery.
Acknowledgements
We would like to thank Mr John Hough for his technical
assistance in preparation of the histological specimens.
Conflict of interest
Dr Torabinejad is currently a consultant to Dentsply
International. In addition, Dr Torabinejad had also a
U.S. patent (Patent No. 8960576 licenced). Dr
Moadde is also on the same patent. The other authors
have stated explicitly that there are no conflict of
interests in connections with this article.
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