Case Report/Clinical Techniques
Histologic Observation of a Human Immature
Permanent Tooth with Irreversible Pulpitis after
Revascularization/Regeneration Procedure
Emi Shimizu, DDS, PhD,* George Jong, DDS,* Nicola Partridge, PhD,† Paul A. Rosenberg, DDS,*
and Louis M. Lin, BDS, DMD, PhD*
Abstract
Introduction: Histological studies of immature human
permanent necrotic teeth with or without apical perio-
dontitis after revascularization have not been reported.
This case report describes the histological findings of
tissue formed in the canal space of an immature perma-
nent tooth #9 with irreversible pulpitis without apical
periodontitis after revascularization. Methods: An
immature human permanent tooth #9 was fractured
3.5 weeks after revascularization and could not be re-
tained. The tooth was extracted and prepared for
routine histological and immunohistochemical evalua-
tion in order to examine the nature of tissue formed in
the root canal following the revascularization procedure.
Results: At 3.5 weeks after revascularization, more
than one half of the canal was filled with loose connec-
tive tissue similar to the pulp tissue. A layer of flattened
odontoblast-like cells lined along the predentin. Layers
of epithelial-like cells, similar to the Hertwig’s epithelial
root sheath, surrounded the root apex. No hard tissue
was formed in the canal. Conclusions: Based on the
histological findings in the present case, regeneration
of pulp-like tissue is possible after revascularization. In
this case, both the apical papilla and the Hertwig’s
epithelial root sheath survived in an immature perma-
nent tooth despite irreversible pulpitis but without
apical periodontitis. (J Endod 2012;38:1293–1297)
Key Words
Immature permanent tooth, irreversible pulpitis, pulp-
like tissue regeneration, revascularization
From the Departments of *Endodontics and †Basic Science
and Craniofacial Biology, New York University College of
Dentistry, New York, New York.
Address requests for reprints to Dr Louis M. Lin, Depart-
ment of Endodontics, New York University College of Dentistry,
345 East 24th Street, New York, NY 10010. E-mail address:
lml7@nyu.edu
0099-2399/$ - see front matter
Copyright ª 2012 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2012.06.017
JOE — Volume 38, Number 9, September 2012
I waya et al (1) showed that a human immature permanent tooth with necrotic pulp
and apical periodontitis/abscess after a revascularization procedure could induce
increased thickening of the canal walls and continued root development. Since
then, many similar cases have been reported (2–5). Radiographically, in some
cases, revascularized human immature permanent teeth appear to show continued
development as evidenced by the deposition of hard tissue on the canal walls and
continued root development. Therefore, the revascularization of human immature
permanent teeth with necrotic pulp and apical periodontitis/abscess has been
considered to be a regenerative process (2–6). Regeneration is defined as the
replacement of damaged tissue by the same parenchymal cells (7). However, regen-
eration is a histologic observation and cannot be determined radiographically. The
nature of the tissue formed in the canal space in human revascularized immature
permanent teeth with apical periodontitis is speculative because no histologic studies
are available.
Currently, the available animal studies of immature teeth with pulp necrosis and
apical periodontitis after revascularization procedures show that the tissues formed in
the canal space are cementoid or osteoid tissue and periodontal ligament–like tissue
(8–11). However, no studies have investigated the nature of the tissue present in
immature teeth with irreversible pulpitis with normal periapical tissues after
a revascularization/regeneration procedure in animals or humans. The purpose of
this case report is to describe the histologic observation of a human immature
permanent tooth clinically diagnosed as having irreversible pulpitis with normal
periapical tissues after a revascularization/regeneration procedure. To our
knowledge, this is the first histologic observation of a human revascularized
immature permanent tooth with irreversible pulpitis.
Materials and Methods
A 10-year-old boy was referred from the Postgraduate Pediatric Clinic at New York
University College of Dentistry to the Postgraduate Endodontic Clinic for the treatment of
tooth #9. The child had a traumatic injury to his maxilla, which caused an uncompli-
cated crown fracture of tooth #8 and a complicated crown fracture with pulp exposure
of tooth #9. The coronal one half of the crown of tooth #9 was horizontally fractured.
According to the patient’s mother, the general dentist removed part of the pulp, placed
medication inside tooth #9, and advised the mother to bring the child to New York
University College of Dentistry for further treatment. The patient and his mother visited
the Postgraduate Pediatric Clinic approximately 1 month after treatment by the general
dentist.
Pulp sensibility tests with Endo-Ice (Coltene/Whaledent Inc, Cuyahoga, OH),
heated gutta-percha, and electric current with the Vitality Scanner (SybronEndo, Glen-
dora, CA) of teeth #8, #9, and #10 were conducted in the Postgraduate Endodontic
Clinic. Teeth #8 and #10 responded to heated gutta-percha, Endo-Ice, and electric
current within normal limits and were not sensitive to palpation or percussion.
The crown of tooth #9 had an Intermediate Restorative Material (Dentsply Internation,
Milford, DE) restoration. It was asymptomatic and responded erratically to pulp sensi-
bility tests because the patient was apprehensive and perhaps also because of the
attempt at pulp therapy performed by the general dentist. The tooth had a large canal
Histologic Observation of an Immature Permanent Tooth 1293
Case Report/Clinical Techniques
space and an open apex surrounded by a well-circumscribed radiolu-
cent area (Fig. 1A). The provisional clinical diagnosis of the pulp-
periapical tissue complex of tooth #9 was pulp necrosis with normal
periapical tissues. A cotton pellet was found in the canal after tooth #9
was accessed after adequate local anesthesia. Bleeding was noted near
the midroot under a Zeiss surgical microscope (Carl Zeiss Meditac
Inc, Dublin, CA). It appeared that a deep pulpotomy or a partial pul-
pectomy might have been previously performed on tooth #9. Conse-
quently, the clinical diagnosis of irreversible pulpitis instead of pulp
necrosis with normal periapical tissues was confirmed. The patient’s
dentist could not be contacted to confirm the prior treatment. It
was not known if a deep pulpotomy or partial pulpectomy was per-
formed under the rubber dam or the pulp had become infected after
deep pulpotomy or partial pulpectomy because the crown was badly
fractured. Therefore, a revascularization/regenerative procedure
instead of apexogenesis was selected for tooth #9 in an attempt to
promote possible pulp tissue regeneration, increased thickening of
the canal walls, and continued root development by Hertwig’s epithe-
lial root sheath (HERS) and apical papilla. The revascularization/
regenerative procedure was performed according to the protocol sug-
gested in our previous study with slight modifications (12). Briefly, at
the first visit, the working length was determined, and the canal was
minimally instrumented with hand K-files (Dentsply Mailefer, Bal-
laigues, Switzerland) and gently irrigated with copious amounts of
5.25% sodium hypochlorite solution (Sultan Healthcare, Hackensack,
NJ) with an irrigation syringe penetrating to the apical portion of the
canal. Calcium hydroxide (Henry Schein, Melville, NY) mixed with
saline solution was used as an interappointment intracanal medication
and carried into the canal with files to apical one third of the canal. At
the second visit 2 weeks later, the tooth was asymptomatic. Under
a surgical microscope, K-files were used to induce bleeding into the
canal by irritating the periapical tissues. A thick mixture of ProRoot
mineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, Tulsa,
OK) and saline solution was used as a coronal seal in the revascular-
ized tooth. The MTA paste was placed into the coronal canal approx-
Figure 1. (A) A preoperative radiograph of tooth #9. (B) A radiograph of the
fractured tooth 3.5 weeks after revascularization. (C) A photograph of the ex-
tracted tooth. Note a small mass of soft tissue attached to the root apex
(arrow). M, mineral trioxide aggregate plug.
1294 Shimizu et al.
imately 5 mm below the access opening against induced bleeding, and
an adequate access cavity was provided for a composite resin restora-
tion. The access cavity was restored with light-cured composite resin
(Amelogen Plus; Ultradent, South Jordan, UT).
Three and a half weeks after completion of the revascularization/
regeneration procedure, the lingual aspect of the crown fractured
below the alveolar crest bone (Fig. 1) and the tooth could not be re-
tained. No pulp sensitivity tests were performed. The tooth was extracted
and processed for histologic and immunohistochemical evaluation. The
crown and MTA plug were removed from the tooth to ensure adequate
penetration of fixative into the canal. The tooth was immediately fixed in
4% formaldehyde for a week and decalcified in 10% EDTA (pH = 7.5)
for 4 weeks at 4 C. The specimen was then soaked in 10% sucrose for 2
hours, in 20% sucrose for 6 hours, and in 30% sucrose overnight and
embedded in optimal cutting temperature compound. Frozen series
sections of approximately 10 mm thickness were cut along the long
axis of the tooth and dried overnight.
Hematoxylin-Eosin Staining
The dried sections were stained with 0.1% Mayer’s hematoxylin
solution for 10 minutes. They were then rinsed in cool running
double-distilled water for 5 minutes, dipped in 0.5 eosin 12 times, dip-
ped in distilled water, and dehydrated in ascending concentrations of
ethanol. The sections were dipped in xylene several times, mounted
on slides, and covered with a coverslip with Cytoseal (Thermo Fisher
Scientific, Waltham, MA) and examined under a light microscope.
Immunohistochemical Staining
To evaluate the localization of mesenchymal stem cells, STRO-1 (R
and D System, Minneapolis, MN) was used. Immunohistochemical stain-
ing was performed using the avidin-biotin complex staining system (Santa
Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s
instructions. The sections were washed in phosphate-buffered saline
(PBS) and immersed in methanol containing 1% hydrogen peroxide
to block endogenous peroxidase activity. The sections were incubated
with 2% blocking serum and then with anti–STRO-1 antibodies or
normal immunoglobulin G as a negative control at 4 C overnight and
washed in PBS. The sections were incubated with biotin-labeled anti–
immunoglobulin G and washed in PBS. Staining was completed by 10
minutes of incubation with 3,3’-diaminobenzidine (Santa Cruz Biotech-
nology, Santa Cruz, CA).
Results
At 3.5 weeks after the revascularization/regeneration procedure of
the immature permanent tooth with clinically diagnosed irreversible
pulpitis, a loose connective tissue with few collagen fibers filled the
canal space up to the coronal MTA plug (Fig. 2A). The tissue in the canal
and periapical tissues were devoid of inflammatory cells. The majority of
cells in the canal space and in the periapical area were spindle-shaped
young fibroblasts or mesenchymal cells. There were more blood vessels
and cellular components in the canal than that at the apical area (Fig. 2B
and C). No nerve-like fibers running alongside the blood vessels were
observed. The tissue in the canal space appeared to be an extension
of the periapical tissue (Fig. 2A). There was a layer of flattened
odontoblast-like cells polarized along the predentin in the apical canal
(Fig. 2B). No hard tissue was seen in the canal space and on the canal
walls. Layers of epithelial-like cells, similar to HERS, surrounded the
root apex (Fig. 2D). Strol-1–positive cells (solid arrows) were observed
in the loose connective tissue near the apical foramen (Fig. 3A and B)
and in the epithelial-like HERS surrounding the root apex. Compared
with the mature dental pulp, the loose connective tissue in the canal
JOE — Volume 38, Number 9, September 2012
 Case Report/Clinical Techniques

Figure 2. (A) Histology of the section of extracted revascularized tooth #9. A loose connective tissue with few collagen fibers has filled the canal space up to the
coronal MTA plug (hematoxylin-eosin, original magnification  200). The MTA plug was removed before histologic tissue processing. (B) High magnification of
the square in A (the apical root canal). Flattened odontoblast-like cells lined along the predentin (solid arrows). Many blood vessels filled with red blood cells
(open arrows). No mature nerve-like bundles along the blood vessels are observed. Most cells are spindle shaped. (C) High magnification of the rectangle in A (the
apical foramen). There are fewer blood vessels (arrow) and cellular components at the apical foramen than that in the canal. (D) High magnification of the square
in C (part of the root apex). Layers of epithelial-like HERS (arrow) surrounding the root apex. Spaces in the tissue are artifacts caused by histologic preparation.

was similar to an immature pulp tissue consisting of numerous spindle-
shaped young fibroblasts or mesenchymal cells, many blood vessels,
few collagen fibers, and no mature nerve-like tissue.
Discussion
It is reasonable to ask why apexogenesis was not attempted in this
case, which was initially diagnosed as an irreversible pulpitis. Irrevers-
ible pulpitis can be caused by trauma, chemical irritation, or bacterial
infection. In this case, bacterial contamination was the major concern
as described previously. If apexogenesis was selected, it was not known
how much of the infected pulp tissue would have to be removed from the
canal. Importantly, apexogenesis in the present case can only
encourage root maturation. It cannot promote pulp tissue regeneration
or ingrowth of vital tissue into the coronal canal space because of a calci-
fied tissue barrier formed in the canal induced by calcium hydroxide or

Figure 3. (A) The immunohistochemistry of the section of extracted revascularized tooth #9 (immunohistochemical staining, original magnification  200). (B)
High magnification of square in A. Strol-1–positive cells in the pulp-like tissue near the apical foramen (arrows) and in the epithelial-like HERS surrounding the
root apex (not shown in high magnification).

JOE — Volume 38, Number 9, September 2012 Histologic Observation of an Immature Permanent Tooth 1295
Case Report/Clinical Techniques
mineral trioxide aggregate in apexogenesis. A revascularization/regen-
eration procedure of immature permanent teeth with irreversible pul-
pitis involving the pulp tissue in the apical portion of the root canal
might have the potential of pulp tissue regeneration into the coronal
canal space, thickening of the canal walls, and continued root develop-
ment because of the survival of HERS and the apical papilla. In addition,
if apexogenesis fails and apical periodontitis develops, the apical papilla
will likely be destroyed, and pulp regeneration will not occur as in
animal studies (8–11).
Based on the present case study, the tissue in the canal space ap-
peared to be an extension from the periapical tissue after the revascu-
larization/regeneration procedure of the immature permanent tooth
with clinically diagnosed irreversible pulpitis. Three and a half weeks
after the revascularization/regeneration procedure, loose connective
tissue filled the canal space up to the coronal MTA plug. The tissue in
the apical canal space was cell rich and well vascularized. This tissue
is similar to cell-rich, well-vascularized connective tissue in the canal
space described by Skoglund and Tronstad (13) in replanted and au-
totransplanted immature teeth of dogs at 30 days of histologic observa-
tions. It is also similar to cell-rich, well-vascularized connective tissue
reported by Claus et al (14) in autotransplanted immature teeth after
removal of the original pulp tissue in beagle dogs at 4 weeks of histo-
logic observations. The loose connective tissue near the apical foramen
contained fewer blood vessels and cell components than the tissue in the
apical canal space. This tissue is similar to the apical papilla described
by Sonoyama et al (15).
The majority of the cells in the periapical tissue and loose connec-
tive tissue in the canal were spindle-shaped young fibroblasts or mesen-
chymal cells. A layer of flattened cells similar to root odontoblasts were
polarized along the predentin in the apical canal. It is not known if these
odontoblast-like cells were preexisting primary odontoblasts or newly
differentiated odontoblasts from the apical papilla after revasculariza-
tion/regeneration procedure of the immature permanent tooth with
irreversible pulpitis. There were few collagen fibers. No nerve-like
fibers running alongside the blood vessels as in mature dental pulp
were observed (16). This may indicate that the loose connective tissue
in the canal is a newly developed immature pulp-like tissue. No hard
tissue was seen in the canal and on the canal walls. Skoglund and Tron-
stad (13) and Claus et al (14) observed hard-tissue formation in the
canal at 30 days and 4 weeks, respectively, after autotransplantation
of dog’s immature teeth. However, Zhao et al (17) observed initial
hard-tissue formation at 7 days after replantation of rat’s immature
teeth. Layers of epithelial-like cells, similar to HERS, surrounded the
root apex, which would regulate continued root maturation (16).
Strol-1–positive stem cells were observed in the loose connective tissue
near the apical foramen. They might be from the apical papilla because
stem cells from the developing apical papilla have greater numbers of
STRO-1–positive cells than stem cells from the mature dental pulp
(15). Mesenchymal stem cells from the apical papilla are capable of
differentiating into odontoblasts under the influence of appropriate
microenvironment cues (15).
There are 2 possible scenarios that could explain the formation of
the pulp-like loose connective tissue in the canal after the revascular-
ization/regeneration procedure in the present case. First, the pulp-like
tissue might be caused by the proliferation of the remaining apical pulp
tissue after the revascularization procedures. However, it is not known
if the residual apical pulp stump in the present case is capable of re-
generating the pulp tissue to fill the coronal canal space after the revas-
cularization/regeneration procedure. Sodium hypochlorite irrigation
and intracanal calcium hydroxide medication might destroy part of
the residual vital apical pulp tissue (18, 19) but not the apical
papilla. Nevertheless, infection is the primary concern and cause of
1296 Shimizu et al.
tissue destruction. It has to be controlled for wound healing
(regeneration or repair) to occur. Second, the pulp-like tissue could
be caused by the proliferation and differentiation of the apical papilla
into the canal space. It has been shown that stem cells from the apical
papilla have more potential than stem cells from the dental pulp to
differentiate into odontoblast-like cells upon receiving appropriate
inductive signals (15, 20, 21). Stem cells from the apical papilla are
derived from a developing tissue that may represent a population of
early stem/progenitor cells that may have a superior cell source for
tissue regeneration (20). Therefore, the pulp-like tissue in the present
case might be derived from the apical papilla. Nevertheless, the rela-
tionship between the apical papilla and the dental pulp needs to be
characterized. If the revascularized tooth in the present case was not
fractured, thickening of the canal walls by deposition of dentin and
continued root development could occur because of the survival of
HERS and the apical papilla and presence of odontoblast-like cells
along the predentin (15, 22).
Conclusion
Based on histologic observation of the present case, regeneration
of the pulp-like tissue is possible after a revascularization/regeneration
procedure because both HERS and the apical papilla survived in an
immature permanent tooth clinically diagnosed as having irreversible
pulpitis. The ideal cases for pulp regeneration in revascularization/
regeneration procedures are likely to be immature permanent teeth
with irreversible pulpitis involving the canal pulp without radiographic
evidence of apical periodontitis or traumatized immature permanent
teeth with a necrotic pulp without radiographic evidence of apical pe-
riodontitis. Apexogenesis is more suitable for irreversible pulpitis
involving only the coronal pulp. Infection and/or inflammation hinder
the potential of tissue regeneration and stem cell function (23). Accord-
ingly, infection/inflammation must be controlled for wound healing
(regeneration or repair) to occur (23).
Acknowledgments
The authors deny any conflicts of interest related to this study.
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