Regenerative Endodontics

Clinical, Radiographic, and Histologic Outcome of

Regenerative Endodontic Treatment in Human
Teeth Using a Novel Collagen-hydroxyapatite
Scaffold
Ali Nosrat, DDS, MS, MDS,*† Alireza Kolahdouzan, DDS, MS,‡§ Amir Hossein Khatibi, DDS, MS,¶
Prashant Verma, DDS, MS, FAGD,† Davoud Jamshidi, DDS, MS,‡ Alan J. Nevins, DDS,k
and Mahmoud Torabinejad, DDS, MSD, PhD**

Abstract
Introduction: Histologic examination of teeth after
regenerative endodontic treatment (RET) shows that
the type, quality, and quantity of tissues formed in the
root canal space are not predictable. The aim of this
study was to examine clinically, radiographically, and
histologically the outcome of RET in immature nonin-
fected human teeth using SynOss Putty (Collagen Matrix
Inc, Oakland, NJ) as a scaffold. Methods: Three pairs of
maxillary/mandibular first premolars in 3 patients sched-
uled for extraction were included. Sensibility tests
confirmed the presence of vital pulps. After informed
consent, anesthesia, and rubber dam isolation, the pulps
were removed. RET was performed using the following
scaffolds: SynOss Putty + blood in both teeth in patient
#1, SynOss Putty with or without blood in patient #2,
and SynOss Putty + blood or blood only in patient #3.
After a follow-up period of 2.5–7.5 months, the teeth
were clinically and radiographically evaluated, ex-
tracted, and examined histologically. Results: Patients
remained asymptomatic after treatment. Radiographic
examination of the teeth showed signs of root develop-
ment after treatment. In teeth treated with SynOss
Putty + blood, histologic examination showed formation
of intracanal mineralized tissue around the scaffold par-
ticles solidifying with newly formed cementumlike tissue
on the dentinal walls. The tooth treated with SynOss
Putty without blood showed the formation of a periap-
ical lesion. The tooth treated with a blood clot only
showed tissues of periodontal origin growing into the
root canal space. Conclusions: SynOss Putty + blood
showed a predictable pattern of tissue formation and
mineralization when used as a scaffold for RET in human immature noninfected teeth.
The newly formed mineralized tissue solidifies with newly formed cementum on the
dentinal walls. (J Endod 2019;45:136–143)
Key Words
Immature tooth, regenerative endodontic treatment, revascularization, revitalization,
scaffold, SynOss Putty
P ulp necrosis in imma-
ture teeth creates a
complicated situation for
Significance
Using SynOss Putty with blood as a scaffold can
make the outcome of regenerative endodontic
clinicians. The immature
treatments more predictable. The formation of an
root/canal is difficult to
intracanal mineralized tissue that solidifies with
disinfect, difficult to seal,
the dentin-associated mineralized tissue might
and prone to fracture
improve the structural integrity of immature teeth.
because of its thin root
structure. Apexification
using mineral trioxide aggregate (MTA) has shown promising results regarding the res-
olution of endodontic disease and the survival of treated teeth (1, 2). Although
traditional apexification methods promote healing of the periapical lesion, they do
not promote root development. Regenerative endodontic treatment (RET) is a
biologically based treatment aiming to regenerate the pulp-dentin complex (3, 4)
and promote continued root development (5). Root development might increase the
overall structural strength of the tooth (6) and, possibly, the survival.
The outcome of RET can be assessed from 2 perspectives:
1. healing of the endodontic disease (ie, successful infection control) and
2. root development (ie, successful tissue regeneration) (7).
As shown in many clinical studies, infection control and healing of the periapical
lesion are predictable and reliable outcomes with this treatment (2, 8, 9). However, the
rate for root development varies from 20% (8) to 100% (10). This means that 1 of the

From the *Iranian Center for Endodontic Research, Dental Research Center, School of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
†
Division of Endodontics, Department of Advanced Oral sciences and Therapeutics, School of Dentistry, University of Maryland Baltimore, Maryland; ‡Department of
Endodontics, School of Dentistry, Ghazvin University of Medical Sciences, Ghazvin, Iran; §Department of Endodontics, School of Dentistry, Shahed University of Medical
Sciences, Tehran, Iran; ¶Private Practice, Tehran, Iran; kPrivate Practice, Southampton, New York; and **Torabinejad Institute of Surgical Education and Research
Venues, Irvine, California.
Address requests for reprints to Dr Ali Nosrat, Division of Endodontics, Department of Advanced Oral sciences and Therapeutics, School of Dentistry, University of
Maryland Baltimore, 650 West Baltimore Street, 4th Floor, Baltimore, MD 21201. E-mail address: Nosrat@umaryland.edu
0099-2399/$ - see front matter
Copyright ª 2018 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2018.10.012

136 Nosrat et al. JOE — Volume 45, Number 2, February 2019


major outcomes of RET is still unpredictable. There are several case re-
ports on “no tissue regeneration” and “empty root canal space” (11,
12) in which teeth treated with RET showed healing of periapical
lesions but no or poor root development. In these cases, the
outcome can be considered a “success” because of infection control
and healing of the periapical lesion or a “failure” because of a lack
of tissue regeneration, evidenced by no root development or an
empty root canal space. Studies have shown that intracanal residual
bacteria play a crucial role in the lack of tissue regeneration in
previously infected immature teeth (13). Other studies hypothesized
that the blood clot might not be stable enough as a scaffold to support
the tissue regeneration process at its initial phases (14). The lack of a
reliable and easy-to-access scaffold is a limitation for the existing pulp
regeneration protocol (15).
Recently, a new scaffold was introduced for RET. SynOss Putty is a
Food and Drug Administration–approved material (https://www.
accessdata.fda.gov/cdrh_docs/pdf7/K072397.pdf) comprising cal-
cium phosphate–based mineral particles with a carbonate apatite struc-
ture combined with bovine type 1 collagen. The mineral particles are
dispersed within collagen fibers forming a 3-dimensional matrix. It is
supplied dry and forms a moldable putty upon hydration. The moldable
putty is durable enough to condense the coronal barrier against it. A
recent case series showed successful results using SynOss Putty as a
scaffold in RET of immature teeth with pulp necrosis and immature teeth
with previous root canal treatment (16). The follow-up radiographs of
those cases showed possible deposition of intracanal mineralized tis-
sues even in previously obturated canals. The findings showed that using
SynOss as a scaffold can lead to a predictable revitalization of the teeth
and production of the intracanal mineralized tissues. There are no his-
tologic data on human immature teeth treated with SynOss Putty. The
aim of this study was to report, for the first time, on the clinical, radio-
graphic, and histologic outcomes of RET in noninfected immature hu-
man teeth using SynOss Putty as a scaffold.
Materials and Methods
Case Selection
The study protocol was peer reviewed and approved by the Insti-
tutional Review Board at the Ghazvin University of Medical Sciences,
Ghazvin, Iran (IR.QUMS.REC.1397.014). The inclusion criteria were
as follows:
1. Bilateral maxillary and mandibular immature premolars planned for
extraction for orthodontic reasons
2. No history of caries or dental procedures
3. A positive response to vitality tests
4. The patient is cooperative enough to conduct the experimental pro-
cedures
5. Parents/guardians are willing to participate
Written informed consent was obtained from the parents. Three
patients were enrolled. Each had a pair of premolars scheduled for
TABLE 1. Demographic Data, Type of Scaffold, and Results of Clinical Examinations at the Recall Sessions
Patient no. Age/sex Tooth no. Scaffold
#1 12/M 20 SynOss + blood
29 SynOss + blood
#2 12/M 5 SynOss + blood
12 SynOss only
#3 10/F 5 SynOss + blood
12 Blood clot only
EPT, electric pulp test; F, female; M, male.
JOE — Volume 45, Number 2, February 2019
Regenerative Endodontics
extraction (Table 1). Preoperative radiographs were taken to confirm
the presence of open apices and the absence of caries and restorations.
In patient #1, teeth #20 and 29 were planned for treatment with SynOss
Putty + blood. The treatments on patient #1 were performed before pa-
tients #2 and 3. After observation of the clinical, radiographic, and his-
tologic outcomes in patient #1, the treatments in patients #2 and 3 were
planned. In patient #2, tooth #5 was planned for treatment with SynOss
Putty + blood, and tooth #12 was planned for treatment with SynOss
Putty only. In patient #3, tooth #5 was planned for treatment with SynOss
Putty + blood, and tooth #12 was planned for treatment with a blood
clot only (Table 1).
Clinical Procedures
After pulp testing using Endo-Ice (Hygenic, Akron, OH) and an
electric pulp tester (Analytic Technology, Redmond, WA), local anes-
thesia was administered using 1.7 mL 3% Carbocaine without epineph-
rine (Novocol Pharmaceutical, Cambridge, Ontario, Canada). Each
tooth was isolated with a rubber dam, and an access cavity was prepared
using a high-speed bur and water spray. After establishing the working
length radiographically 1 mm short of the open apex, the coronal thirds
of the canals were enlarged using Gates Glidden drills (Dentsply Mail-
lefer, Ballaigues, Switzerland). All canals were instrumented using hand
files (Dentsply Maillefer) to completely remove the pulp tissue. Between
each instrument, the canals were gently irrigated with 1.25% sodium
hypochlorite at 1 mm short of the working length. After completion
of instrumentation, all canals were irrigated with 5 mL 17% EDTA
and dried with sterile paper points. Bleeding was induced by overexten-
sion of a size 30 hand file 2–3 mm beyond the working length. In teeth
receiving SynOss Putty + blood as a scaffold, the material was cut into
several pieces, soaked in normal saline, placed in the root canal orifice,
and packed apically to the working length using a 5/7 plugger (1 piece
at the time). The procedure was performed immediately after bleeding
was induced. A blood clot was then allowed to form for 10 minutes. MTA
powder and liquid (ProRoot; Dentsply Tulsa Dental, Tulsa, OK) were
mixed to putty consistency, gently placed over the blood clot/SynOss
Putty + blood, and gently adapted to the dentinal walls. Because SynOss
Putty creates a firm surface in the root canal space, MTA could be easily
packed against it. The access cavity of each tooth was restored with
resin-modified glass ionomer cement. A postoperative periapical radio-
graph was taken. There was insufficient bleeding induced in tooth #5 in
patient #2. The blood only filled the apical third of the canal. The rest of
the procedures were performed as planned.
The length of the recall period before extraction was determined
by the orthodontic treatment plan. Before extraction, a periapical radio-
graph was taken to evaluate the continuation of root development. Pulp
sensibility tests were performed, and the results were recorded. The
teeth were extracted under local anesthesia. Immediately after extrac-
tion, 2 radiographs were taken of each tooth (the mesiodistal view
and the buccolingual view).
Recall (months) Percussion Palpation EPT Cold test
7.5 — — + +
2.5 — — — —
3 — — — —
3 — — — —
4 — — — —
4 — — — —
Regenerative Endodontic Treatment Using SynOss Putty Scaffold 137
Regenerative Endodontics
Tissue Processing
The teeth were placed in 10% formaldehyde for fixation.
Teeth extracted from patient #2 had a narrower apical opening,
so small round holes were prepared on the roots to ensure com-
plete diffusion of formaldehyde into the canal spaces. All teeth
were decalcified in 7% formic acid. Complete decalcification was
confirmed radiographically. After decalcification, specimens were
rinsed with running tap water for 2 hours and dehydrated with
ascending concentrations of alcohol (70%, 90%, and 100%).
The glass ionomer restorations were gently removed, and speci-
mens were embedded in paraffin. Five-micrometer-thick labiolin-
gual serial sections were prepared and stained with hematoxylin-
eosin. Masson trichrome staining was performed on sections
from tooth #20 in patient #1. Samples were evaluated microscop-
ically (Zeiss, G€ottingen, Germany) by an independent oral pathol-
ogist to determine the histologic features of tissues formed within
the root canal spaces.
Results
All enrolled teeth responded positively to sensibility tests before
the procedures. None of the patients reported postoperative pain or
discomfort. The demographic data and recall periods are presented
in Table 1. The longest recall period was 7.5 months for tooth #20 in
patient #1 (SynOss Putty + blood). The shortest recall period was
2.5 months for tooth #29 in patient #1 (SynOss Putty + blood)
(Table 1). The results of the clinical, radiographic, and histologic ex-
aminations are as follows.
Clinical Findings
All treated teeth were asymptomatic and functional at recall ses-
sions, with normal mobility, normal probing depths, and no signs or
symptoms of endodontic disease. The teeth responded negatively to sen-
sibility tests at the recall session, except for tooth #20 in patient #1 (Syn-
Oss Putty + blood), which responded positively to both the cold test and
electric pulp testing at the 7.5-month recall. Details of the clinical find-
ings at the recall sessions are shown in Table 1.
Radiographic Findings
In the radiographs taken before extraction, both mandibular pre-
molars in patient #1 (SynOss Putty + blood) showed variable degrees of
root development with apical closure and increased opacity in canal
spaces (Fig. 1A–J). The maxillary premolars in patient #2 (SynOss
Putty + minimal blood in tooth #5 and SynOss Putty only in tooth
#12) showed no changes in root dimensions or opacity in canal spaces
(Fig. 2A–J), except apical closure in tooth #5 (Fig. 2H). Both maxillary
premolars in patient #3 (SynOss Putty + blood in tooth #5 and blood
only in tooth #12) showed root development (Fig. 3A–J), with increased
opacity in the coronal and middle thirds only observed in tooth #5 (Syn-
Oss Putty + blood) (Fig. 3H).
The radiographs after extraction showed that SynOss Putty was
successfully placed into the entire root canal system in both teeth in pa-
tient #1 (SynOss Putty + blood) (Fig. 1D, E, I, and J) and in both roots of
tooth #5 (SynOss Putty + minimal blood) (Fig. 2I and J) and the palatal
root of tooth #12 (SynOss Putty only) (Fig. 2D and E) in patient #2.
Tooth #5 in patient #3 (SynOss Putty + blood) showed an opaque canal,

Figure 1. Radiographic images of patient #1. (A) The preoperative radiograph of tooth #20 treated with SynOss Putty + blood. (B) The postoperative radiograph of
tooth #20. The increased opacity is because the canal was filled with SynOss Putty. (C) The 7.5-month follow-up radiograph shows increased opacity in the root
canal space and formation of the apex. Postextraction (D) buccolingual and (E) mesiodistal views, respectively. Note the increased opacity from apical to coronal.
(F) The preoperative radiograph of tooth #29 treated with SynOss Putty + blood. (G) The postoperative radiograph of tooth #29. The increased opacity is because
the canal was filled with SynOss Putty. (H) The 2.5-month follow up radiograph. Postextraction (I) buccolingual and (J) mesiodistal views. Note the increased
opacity from apical to coronal.

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Figure 2. Radiographic images of patient #2. (A) The preoperative view of tooth #12 treated with SynOss Putty only. (B) The postoperative radiograph of tooth
#12. The increased opacity is because the canal was filled with SynOss Putty. (C) The 3-month follow-up. Postextraction (D) buccolingual and (E) mesiodistal
views. Note the apices are still open, and SynOss Putty did not reach the apical third in the buccal canal. (F) The preoperative radiograph of tooth #5 treated with
SynOss Putty + blood. Bleeding was insufficient and only filled the apical third. (G) The postoperative radiograph of tooth #5. The increased opacity is because the
canal was filled with SynOss Putty. (H) The 3-month follow-up shows the formation of apices. Postextraction (I) buccolingual and (J) mesiodistal views.

except in the apical 2 mm (Fig. 3I and J). Tooth #12 in patient #3 (blood
clot only) showed intracanal opacity limited to a narrow zone below
MTA (Fig. 3D and E). All extracted teeth had immature apices with var-
iable openings as shown in the buccolingual views. Comparison of the
MTA level between these teeth showed well-controlled placement in all
SynOss Putty cases versus poor control (ie, apical displacement) in the
blood clot case.
Histologic Evaluations
Hematoxylin-eosin Staining
Teeth #20 (Fig. 4A–G) and #29 (Fig. 5A–C) in patient #1 and tooth
#5 in patient #3 (in the coronal and midroot areas) (Fig. 5G and H)
showed a similar pattern of new tissue formation. Normal root anatomy
was noted with no resorption in the dentin and no inflammation in the
newly formed intracanal tissues. The newly formed tissue contained
normal connective tissue, fibroblasts, and blood vessels. There were
2 types of newly formed mineralized tissue: a cellular intracanal miner-
alized tissue that was associated with the scaffold particles and a cellular
cementumlike tissue associated (ie, fused) with dentinal walls, which
showed scaffold particles entrapped in some areas (Fig. 4B–D). The
cells were entrapped in small lacunae within the matrix in both miner-
alized tissues. In some areas, the newly formed intracanal mineralized
tissue showed no cell entrapment within the structure. Several areas of
fusion and calciotraumatic lines between these mineralized tissues were
observed. This newly formed tissue was continuous with the periodontal
ligament at the apex, where the scaffold particles were absent. The nar-
rowing at the apex was caused by formation of the newly formed cellular
cementumlike tissue (Fig. 4F and G). There were few bony ingrowths
into the root canal spaces (Fig. 4E).
The newly formed intracanal mineralized tissue was denser and
occupied most of the space with less connective tissue in the coronal
third (below the MTA) and the middle third compared with the apical
third (Figs. 4A and 5G and H). Tooth #29 in patient #2 had the shortest
follow-up time (2.5 months) and showed early phases of remodeling of
the scaffold and minimal mineralized tissue formation compared with
tooth #20 in the same patient (Figs. 5A–C compared with Fig. 4). No
pulp tissue (characterized by the presence of odontoblastlike cells
aligning root canal walls) was observed in any of the sections in these
teeth. There was no intracanal or periapical inflammation in any of the
samples.
The sections from the tooth treated with SynOss Putty without
blood (patient #2, tooth #12) showed no tissue formation in the root
canal spaces of both roots (Fig. 5F). There was periapical inflammation
extending slightly into the root canal lumen through the apical foramen.
Several resorptive lacunae were observed in the dentinal walls (Fig. 5F).
No predentin was found in these sections. The midroot and coronal sec-
tions of the root canal spaces were filled with what appeared to be dis-
integrating SynOss Putty particles (Fig. 5F).
The sections from the tooth treated with SynOss Putty and minimal
bleeding (limited to the apical third) showed newly formed tissues
similar to other teeth treated with SynOss Putty + blood only in the apical
area (Fig. 5E). The rest of the root canal spaces were filled with disinte-
grating SynOss Putty particles (Fig. 5E). No inflammation or resorption
was found in this tooth.
The sections from the tooth treated with a blood clot (patient #3,
tooth #12) revealed portions of roots showing well-developed dentin. A

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Figure 3. Radiographic images of patient #3. (A) The preoperative radiograph of tooth #12 treated with a blood clot only. (B) The postoperative radiograph of
tooth #12. (C) The 4-month follow up shows continued root development. Postextraction (D) buccolingual and (E) mesiodistal views, respectively. Note the for-
mation of a radiopaque barrier underneath MTA. (F) The preoperative radiograph of tooth #5 treated with SynOss Putty + blood. (G) The postoperative radiograph
of tooth #5. The increased opacity is because the canal was filled with SynOss Putty. (H) The 4-month follow-up of tooth #5 shows increased opacity in the coronal to
middle third and continued root development. Postextraction (I) buccolingual and (J) mesiodistal views. Note the increased opacity from apical to coronal.

Figure 4. Hematoxylin-eosin staining of the apical third of tooth #20 in patient #1 (SynOss Putty + blood). (A) Lower magnification (40) shows the newly
formed connective/mineralized tissues in the root canal space. (B) Higher magnification (100) of the area showing integration among the newly formed miner-
alized tissues on the dentinal walls, the newly formed mineralized tissue in the root canal space, and the scaffold particles. (C and D) Higher magnification (200)
of the cellular dentin–associated mineralized tissue (DAMT) and particles of scaffold within or in close contact (arrows) with DAMT, showing remodeling of the
scaffold. (E) A view of a bony island (BI) formed close to the apical foramen (100). (F and G) A view of DAMT at the apex (200). d, dentin; c, cementum; S,
scaffold particle.

140 Nosrat et al. JOE — Volume 45, Number 2, February 2019

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Figure 5. (A) Hematoxylin-eosin staining of tooth #29 in patient #1 (SynOss Putty + blood) showing the loose connective tissue mixed with a scaffold (40). (B)
The left area outlined in A. A scaffold particle integrated with connective tissue showing early phases of remodeling. (C) The right area outlined in A. Newly formed
mineralized tissue intermixed with scaffold particles and connective tissue (100). (D) Masson trichrome staining of tooth #20 in patient #1 (SynOss
Putty + blood) (40). The blue color represents collagen fibers with a high concentration in intracanal tissues and scaffold particles. (E) Tooth #5 in patient
#2 (SynOss Putty + minimal blood) showing tissue formation only in the apical few millimeters (20). (F) Tooth #12 in patient #2 (SynOss Putty only) showing no
tissue formation, intracanal and periapical inflammation, resorptive lacunae (arrows), and disintegrating SynOss in the root canal space. (G) The coronal third in
tooth #5 of patient #3 (SynOss Putty + blood) (100). The newly formed intracanal mineralized tissue is intermixed with scaffold particles and connective tissue.
This is an area of well-condensed SynOss Putty mixed with blood that is transitioning toward full calcification. (H) The midroot of tooth #5 (SynOss Putty + blood) in
patient #3 (200) shows areas of solidification between dentinal walls, DAMT, and the newly formed intracanal mineralized tissue with calciotraumatic lines in
between. (I) The coronal third of tooth #12 of patient #3 (blood only) showing the formation of intracanal osteoidlike tissue (OSLT) and a cementumlike tissue on
the dentinal walls (100). (J) The midroot of tooth #12 of patient #3 (blood only) showing formation of the cementumlike tissue (DAMT) on the dentinal walls and
loose connective tissue (CT) in the root canal space (200). d, dentin; S, scaffold particle; pd, predentin.

well-preserved primary dentin layer was identified surrounding the ca-
nal spaces (Fig. 5I and J). Foreign body material (MTA) was also
observed in the coronal portions of this tooth (Fig. 5I). A thick layer
of hard tissue was formed underneath the MTA in this specimen. There
is a fibrotic connective tissue within the canal spaces (Fig. 5J) and oc-
casional globular and malformed cementum and dystrophic calcifica-
tion (Fig. 5I and J). The root canal walls are covered with a well-
formed symmetric layer of reparative cementum admixed with osteoid
tissue (Fig. 5J). No pulp tissue (characterized by the presence of odon-
toblastlike cells aligning root canal walls) was noted in any of the sec-
tions in this tooth. There was no intracanal or periapical inflammation.
Masson Trichrome Staining
The blue color highlighting collagen was observed in dentin,
dentin-associated newly formed mineralized tissue, intracanal newly
formed mineralized tissue, newly formed connective tissue, and the
periodontal ligament. Also, scaffold particles are stained blue, indi-
cating the presence of collagen (Fig. 5D).
Discussion
To determine the potential outcome of a tissue engineering strat-
egy in RET, it is logical to first test it in a noninfected model. The reason
is that the known negative effects of disinfection processes on the root
canal microenvironment (17) and stem cells (18, 19) and the negative
effects of residual bacteria on newly formed tissues (13) will be
excluded from the experiment. In our previous study, we used imma-
ture human premolars planned for orthodontic extractions to test the
outcome of RET in noninfected root canal systems (20). The study es-
tablished a reliable noninfected model to examine the histologic out-
comes of tissue engineering strategies for RET. Also, using a human
model is favored over animal models because of differences in the bio-
logical dynamics (21).
The histologic outcomes of RETs using different tissue engineering
approaches have been extensively studied. The effects of different com-
binations of dental pulp stem cells, collagen scaffolds, platelet-rich
plasma, platelet-rich fibrin, and growth factors have been examined
in animal models (22–24). None of these tissue engineering
strategies resulted in true pulp-dentin complex regeneration. The
main histologic findings in these studies were the formation of bone/
bonelike islands in the root canal space and deposition of a mineralized
cementumlike tissue on the dentinal walls. Some studies described
these findings as “ingrowth” of the periodontium into the root canal
space (25). Furthermore, many of these animal studies showed that
the quantity of newly formed tissues in the root canal space is unpredict-
able. The newly formed tissues were limited to the apical third of the
canal as shown in several animal studies (13, 26).
The unpredictable histologic outcome in animal studies explains
the findings of “poor or no root development” (11, 14, 27, 28) or
“empty root canal” (11, 12, 14) in several clinical studies. No root
development can be an indication of poor or no tissue regeneration
after treatment. The histologic findings in animal studies are
consistent with findings in human teeth when a blood clot (20) or
platelet-rich plasma (29) was used as a scaffold. This unpredictability
of tissue formation after RET can be partly because of suboptimal scaf-
folds used in current protocols.
Overall, it seems that “true pulp regeneration” is out of reach at
this point and that existing protocols result in an unpredictable outcome
regarding the type, quality, and quantity of the newly formed tissues. The
present study showed that using SynOss Putty as a scaffold can change
this dynamic. The teeth treated with SynOss Putty + blood showed a
consistent pattern in the formation of new tissues. This scaffold provides

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a matrix for the formation of a mineralized tissue that solidifies with the
newly formed cementumlike tissue on the dentinal walls. Perhaps it
makes the entire root canal space calcified over time, as documented
radiographically in the previous clinical study (16). This outcome is
very similar to pulp canal obliteration in immature teeth after dental
trauma. The origin of the newly formed intracanal mineralized tissue
is not determined in this study. This subject deserves further investiga-
tions.
A recent study on the biomechanical properties of immature teeth
that had undergone RET showed that these teeth have a higher fracture
resistance compared with immature teeth with pulp necrosis and no
treatment (6). This indicates that root thickening caused by cementum
formation on dentinal walls does strengthen the root structure. Several
histologic studies in animal and human teeth have shown that this
cementum formation happens to variable degrees (length and thick-
ness) on dentinal walls, and the root canal space is filled with variable
quantities of bony islands and loose connective tissue. The teeth treated
with SynOss Putty + blood showed the formation of mineralized tissue in
the root canal space fusing with the newly formed cementumlike tissue
on the dentinal walls. The histologic differences observed between teeth
treated with a blood clot only and teeth treated with SynOss
Putty + blood (Fig. 5G and H compared with I and J) imply that the latter
might have higher fracture resistance because of better physical integ-
rity. However, this is a speculation based on histologic observations in
few teeth. Further research in this area is recommended.
The present study shows that blood plays a crucial role in the for-
mation of the new tissues in the root canal space when SynOss Putty is
used as a scaffold. The tooth treated with SynOss Putty without blood
showed no tissue regeneration with periapical and intracanal inflamma-
tion. When the blood level was limited to the apical third (tooth #5, pa-
tient #2), the tissue formation was also limited to the apical third. Blood
originating from periapical tissues contains a very high concentration of
mesenchymal stem cell markers (up to 600 times) compared with pe-
ripheral blood (30). The blood clot is a protein-rich scaffold containing
platelet-derived growth factors and signaling molecules (31). These
growth factors and signaling molecules promote proliferation and dif-
ferentiation of stem cells. Therefore, SynOss Putty should be used with
blood to perform as a scaffold in RET.
The tooth treated with a blood clot only showed radiographic and
histologic outcomes very similar to those reported in previous studies
(20). The root development observed radiographically was caused by
the formation of cementumlike tissue on the dentinal walls. A blood
clot has been used as a physical scaffold in many case reports (4, 32).
One animal study showed that RETs with blood clot formation had a
better radiographic outcome compared with those without blood clot
formation (33). Lack of a blood clot was associated with poor root devel-
opment in clinical case reports (4, 11). The results of the present study
show that SynOss Putty + blood provides a more predictable outcome
compared with a blood clot only. On the other hand, the follow-up period
in the present study was short. Further remodeling of intracanal tissues
during years after RET can make significant changes in the histologic out-
comes. Further clinical studies with longer follow-ups are recommended
to confirm the findings of this study.
As in many previous studies, MTA was used as a coronal barrier
over the scaffold (12, 34). MTA is a bioactive material that produces
hydroxyapatite crystals when in contact with body fluids (35). MTA is
biocompatible, promotes cell differentiation, and induces hard tissue
formation without adverse tissue reactions (36). The sealing ability of
MTA makes it a suitable biomaterial for use as an orifice barrier to pre-
vent bacterial ingress over time (36). There was no difficulty in the
placement of MTA and controlling the level of MTA in teeth treated
with SynOss Putty + blood as this scaffold forms a firm surface.
142 Nosrat et al.
In conclusion, SynOss Putty + blood showed a predictable pattern
of tissue formation and mineralization when used as a scaffold for RET
in human noninfected teeth. The newly formed mineralized tissue solid-
ifies with the newly formed cementum on the dentinal walls. Further
clinical studies on previously infected teeth with larger sample sizes
and longer recall periods are recommended to determine the efficacy
of this novel scaffold on the success and survival rates of RET.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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