Regenerative Endodontics
Survival of the Apical Papilla and Its Resident
Stem Cells in a Case of Advanced Pulpal Necrosis
and Apical Periodontitis
Vanessa Chrepa, DDS, MS,*† Brandon Pitcher, DDS,† Michael A. Henry, DDS, PhD,†‡
and Anibal Diogenes, DDS, PhD†
Abstract
Introduction: Apical papilla represents a source of an
enriched mesenchymal stem cell (MSC) population
(stem cells of the apical papilla [SCAPs]) that modulates
root development and may participate in regenerative
endodontic procedures in immature teeth with pulp ne-
crosis. The characteristics and phenotype of this tissue in
the presence of inflammation are largely unknown. The
purpose of this study was to characterize a human apical
papilla sample that was isolated from an immature
tooth with pulp necrosis and apical periodontitis.
Methods: Inflamed periapical tissue that included
part of the apical papilla (apical papilla clinical sample
[CS]) was collected from an immature mandibular pre-
molar previously diagnosed with pulp necrosis and api-
cal periodontitis during an apexification procedure.
Harvested cells from this tissue (SCAP CS) were
compared with inflamed periapical progenitor cells
(IPAPCs) and normal SCAP (SCAP-RP89) in flow cytom-
etry and quantitative osteogenesis experiments. Part of
the issue was further processed for immunohistochem-
istry and compared with apical papilla and coronal
pulp sections from normal immature teeth as well as in-
flamed periapical tissues from mature teeth. Results:
Similar to SCAP-RP89, 96.6% of the SCAP CS coex-
pressed the MSC markers CD73, CD90, and CD105,
whereas only 66.3% of IPAPCs coexpressed all markers.
The SCAP CS showed a significantly greater mineraliza-
tion potential than both SCAP-RP89 and IPAPCs. Finally,
immunohistochemical analysis revealed moderate infil-
tration of cells expressing the inflammatory markers
CD45/68 in the apical papilla CS and prominent CD24,
CD105, and von Willebrand factor expression. Conclu-
sions: Under inflammatory conditions, human apical
papilla was found moderately inflamed with retained
SCAP vitality and stemness and increased osteogenic
and angiogenesis potential. (J Endod 2016;-:1–7)
From the *Department of Endodontics, University of Washington, Seattle, Washington; †Department of Endodontics, University of Texas Health Science Center at
San Antonio, San Antonio, Texas; and ‡University of Colorado School of Dental Medicine, Aurora, Colorado.
Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive,
San Antonio, TX 78229. E-mail address: Diogenes@uthscsa.edu
0099-2399/$ - see front matter
Copyright ª 2016 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.09.024
JOE — Volume -, Number -, - 2016
Key Words
Apical papilla survival, apical periodontitis, characterization, periapical inflammation,
regenerative endodontics, stem cell, stem cells of the apical papilla
T he apical papilla con-
sists of the apical portion
of the dental papilla and,
Significance
The apical papilla may survive despite intense
inflammatory infiltrate following pulp necrosis. In
in conjunction with the
this report, SCAPs maintained their stemness and
Hertwig epithelial root
expressed increased osteogenic and angiogenesis
sheath, is responsible for
potential. Regenerative strategies should focus on
root development (1). Stem
promoting the continued survival, recruitment, and
cells of the apical papilla
differentiation of these cells to achieve predictable
(SCAPs) have been shown
guided endodontic repair and regeneration.
to have great proliferation
and differentiation poten-
tial in addition to high motility (2). Studies have highlighted the potential role of SCAPs
and the apical papilla in the continuation of root development and regeneration of the
pulp-dentin complex (1, 3). Notably, in a pilot experiment, surgically removing the
apical papilla resulted in the arrest of root development even in the presence of
intact pulp (1). Huang et al (3) further showed that SCAPs have the ability to differen-
tiate into odontoblastlike cells and lead to de novo dental pulp regeneration in vivo.
These findings suggest the importance of maintaining the vitality of the apical papilla
in immature teeth as a source of stem cells that contribute to and regulate root devel-
opment.
In regenerative endodontic procedures (REPs), evoked bleeding from the periap-
ical tissues has been shown to lead to a significant influx of mesenchymal stem cells
(MSCs) in the root canal system of both immature and mature teeth (4, 5). Using
this step as a method to introduce stem cells into the root canals is a significant
concept in regenerative endodontics because it provides access to the most readily
available sources of MSCs (ie, apical papilla, periodontal ligament, alveolar bone,
and inflamed periapical tissues) for potential dental pulp regeneration (6). In imma-
ture teeth, the apical papilla represents an enriched pool of MSCs in direct contact with
the tooth apex (2, 7). Even with the odontogenic differentiation potential of SCAPs, REPs
do not always result in the formation of dentin and pulplike tissue (8–10). The root
canal microenvironment including pulp status and infection control regimens seems
to affect the regeneration phenotype (8, 11–16). REPs in teeth with pulp necrosis
and harboring bacteria in the root canal system have been associated with
Survival of the Apical Papilla 1
Regenerative Endodontics
cementum or bonelike tissue formation, whereas dentin formation was
noted in teeth with irreversible pulpitis (11, 12). Importantly,
prolonged infection and periapical inflammation may seriously affect
the survival of the apical papilla or alter its properties, which is
considered crucial for the continuation of root development in
immature teeth that undergo regenerative endodontic treatment (1).
Despite the importance of the latter, the effect of pulp necrosis and peri-
apical inflammation on the survival of human apical papilla is largely
unknown.
Studies have identified unique properties and characteristics
of the human apical papilla and SCAPs; however, the included sam-
ples were from human immature teeth with normal pulp and peri-
apical tissues (2, 3, 17, 18). These studies showed the distinct
histologic and immunohistochemical properties of apical papilla
compared with dental pulp as well as characterized SCAP cell
lines in terms of gene expression, stemness, and differentiation
potential (2, 3, 17, 18). Nonetheless, it is very likely that apical
papilla and SCAPs present differently in pulp necrosis and
periapical inflammation (1). Hypoxia and inflammation have
been shown to affect SCAP proliferation and differentiation in cul-
ture (19, 20). Tobias Duarte et al (21) evaluated the histopatho-
logical conditions of dental pulp and apical papilla after inducing
endodontic infection in a rat model and found that apical papilla
remained vital after 90 days of infection yet slightly or moderately
inflamed. Despite the current evidence, to date, no study has eval-
uated human apical papilla from an immature tooth with pulp ne-
crosis and apical periodontitis. The purpose of this study was to
report for the first time the characterization of a human apical
papilla sample harvested through the canal system of an immature
tooth with pulp necrosis being treated with an apexification pro-
Figure 1. Case report radiograph and clinical photographs. (A) A periapical radiograph of tooth #28. (B) Periapical tissue was engaged to a H-file after removal
from the root canal of tooth #28. (C) Tissue was immediately sectioned into 2 pieces, both containing part of the apical papilla (green arrow) and part of the
inflamed periapical tissue (black arrow). The yellow lines represent the junction between the 2 distinct tissues.
2 Chrepa et al.
cedure. We hypothesized that apical papilla and its resident cells
retain their vitality under chronic inflammatory conditions.
Materials and Methods
Case Report
A 9-year-old female presented to the Graduate Endodontics Clinic at
the University of Texas Health Science Center at San Antonio, San Antonio,
TX, for evaluation and treatment of tooth #28. The patient reported tooth
sensitivity in the past, approximately 1 year before the appointment, pro-
duced by hot/cold drinks, and her complaint at the time of the visit was
sensitivity on biting. Tooth #28 presented clinically with extensive
occlusal caries, normal probing depths and mobility, and normal soft tis-
sues. Radiographically, tooth #28 presented with a carious lesion extend-
ing into the pulp chamber, immature apex, and periapical radiolucency
(Fig. 1A). Pulp sensibility tests with Endo-Ice (Coltene/Whaledent Inc,
Cuyahoga, OH) and an electric pulp test (Electric Pulp Tester; Sybro-
nEndo, Glendora, CA) were performed, and negative pulp responses
were noted. Periradicular tests revealed that tooth #28 was sensitive to
percussion but not sensitive to palpation. Based on the clinical tests,
the pulpal diagnosis was determined as pulp necrosis and the periradic-
ular diagnosis as symptomatic apical periodontitis. Both the guardian
and patient were informed of the clinical findings and presented the treat-
ment options of regenerative endodontic treatment, apexification with a
mineral trioxide aggregate (MTA) plug, extraction, and no treatment. The
guardian and patient elected MTA apexification and signed the informed
consent. After adequate anesthesia, the tooth was isolated with a rubber
dam, and access was made into the root canal system. Pulp necrosis was
confirmed clinically upon access. For the chemomechanical prepara-
tion, Hedstrom files (H-files, SybronEndo) were used to file the canal
JOE — Volume -, Number -, - 2016

walls along with 6% sodium hypochlorite irrigation. During the mechan-
ical preparation, tissue from the periapical region was removed with a
size 25 H-file placed 2 mm beyond the apex. The tissue was engaged
with the H-file and removed from the canal system while still attached
to the file (Fig. 1B). After careful inspection with the use of 10 magni-
fication, we confirmed that this tissue included part of the apical papilla
and part of the inflamed periapical tissues (Fig. 1B). With the guardian’s
consent, this tissue, which would have otherwise been discarded, was
collected immediately in cold Hank’s balanced salt solution (Invitrogen,
Carlsbad, CA) for processing. At the end of the first appointment,
Ca(OH)2 was placed as the interim intracanal medication. The patient
presented asymptomatic at the second visit, and MTA apexification was
completed. The guardian was informed that the tooth prognosis was
favorable, and subsequent follow-ups were scheduled.
Sample Collection and Processing
The apical papilla clinical sample (CS) was transferred immediately
to the laboratory where it was sectioned into 2 pieces (Fig. 1C). One piece
was processed for immunohistochemistry and confocal microscopy as
previously described in order to detect antigens in the tissue specific
for stem cells and inflammatory cells (18). The other piece was used
to generate a SCAP culture of the CS and contained the apical papilla
(Fig. 1C, green arrow), which was clearly distinct from its attached apical
tissue (Fig. 1C, black arrow). The apical papilla was carefully dissected
from the rest of the tissue with a sterile 15C blade, separating it at the level
of junction between the 2 distinct tissues (Fig. 1C, yellow lines). The
dissected apical papilla tissue was minced, subjected to enzymatic diges-
tion, and processed for cell culture as described previously (16). Har-
vested cells were cultured at 37 C and 5% CO2 in basal culture
medium composed of alpha-minimum essential medium (Gibco, Grand
Island, NY) supplemented with 10% fetal bovine, L-glutamine (Gemini,
West Sacramento, CA), penicillin (100 U/mL), streptomycin (100 mg/
mL), and amphotericin B (Sigma-Aldrich, St Louis, MO). Single-cell col-
onies were transferred to 10-cm culture dishes, maintained, and cultured
until 80% confluency. Cells in the 5th passage were used for flow cy-
tometry and differentiation experiments.
For immunohistochemical processing, the tissue (ie, apical papilla
CS) was rinsed 3 times (5 minutes each) in 0.1 mol/L phosphate buffer
(PB) followed by fixation in 4% paraformaldehyde in 0.1 mol/L PB and
then stored at 20 C for 24 hours in 0.1 mol/L PB with 30% sucrose.
The tissue was then serially sectioned with a cryostat (30-mm sections);
sections were collected onto glass slides and stored at 20 C until ready
for staining. Sections from the apical papilla CS tissue sample that was
processed for immunohistochemistry included part of the inflamed tis-
sue and part of the apical papilla (Fig. 1C, black and green arrows,
respectively). To better assess the phenotype of these cells (ie, SCAP
CS) and their source tissue (ie, apical papilla CS), additional cell lines
and tissue slides previously collected, processed, and stored in our de-
partment’s tissue bank were included in the analysis. The cell lines
included were inflamed periapical progenitor cells (IPAPCs) previously
harvested from a periradicular lesion of an adult patient and a character-
ized SCAP cell line (ie, SCAP-RP89) from a young donor (18). These cell
lines were used in the flow cytometry and differentiation experiments
along with SCAP CS. The tissue slides included apical papilla and coronal
pulp sections from normal immature teeth (n = 2) as well as biopsies of
inflamed periapical tissues collected during apicoectomies in adult pa-
tients and diagnosed as cysts or granulomas (n = 5) (4). These tissue
slices were processed for immunohistochemistry along with apical
papilla CS slides. The Institutional Review Board of the University of Texas
Health Science Center at San Antonio had approved the collection of these
tissues and cell lines for previous studies (4, 16, 18).
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Regenerative Endodontics
Flow Cytometry
Single-cell suspensions of SCAP CS and IPAPCs (106 cells/mL) were
incubated in Stain Buffer (FBS; BD Biosciences, San Jose, CA) concomi-
tantly with fluorescent-labeled antibodies and processed for multicolor
flow cytometry analysis as previously described (4). The following anti-
bodies were included: PE-CY7 mouse antihuman CD73 (561258, BD Bio-
sciences), APC mouse antihuman CD90 (clone 5E10, BD Biosciences),
PerCP-CY5.5 mouse antihuman CD105 (clone 266RUO, BD Biosciences),
and APC-H7 mouse antihuman CD45 (clone 2D1BD, Biosciences). Gating
was performed on CD45-negative cells and included doublet discrimina-
tion for single cells and scatter gate for gating out small debris. For each cell
line, the percentage of cells expressing each marker and the percentage of
the cells coexpressing CD73, CD90, and CD105 was recorded. Results
were compared with previously published flow cytometry data including
the same MSC markers for the cell line RP-89 (SCAP-RP89) (18).
Alizarin Red Staining and Osteogenesis Quantification
A previously characterized SCAP cell line (SCAP-RP89), SCAP CS,
and IPAPC cell lines (5th passage for each) were cultured in 24-well
plates containing either basal medium (control) or osteogenesis induc-
tion medium (StemPro Osteogenesis Differentiation Kit; Life Technolo-
gies, Grand Island, NY) for 3 weeks (n = 6/cell line). Qualitative and
quantitative analysis of mineralization was then performed using the
Osteogenesis Quantitation Kit (Millipore, Darmstadt, Germany)
following the manufacture’s protocol. Images of the alizarin red staining
were taken under light microscopy.
Immunohistochemistry and Laser Confocal Microscopy
Tissue slices from the apical papilla CS, apical papilla (normal pulp),
coronal pulp (normal pulp), and inflamed periapical lesions were pro-
cessed for immunohistochemistry as previously described (22). Immuno-
histochemical analysis included the primary antibodies CD105 (mouse
antihuman, M3527, 1:500 dilution; Dako, Glostrup, Denmark), CD24
(mouse antihuman, 555426, 1:500 dilution; BD Biosciences), CD45
(mouse antihuman, M0701, 1:200 dilution; Dako), and CD68 (mouse
antihuman, M0876, 1:200 dilution; Dako) combined with the endothelial
cell marker von Willebrand factor (vWF) antibody (rabbit antihuman,
A0082, 1:4,000 dilution; Dako). Goat antimouse Alexa Fluor 488 and
goat antirabbit Alexa Fluor 568 (both antibodies at 1:100 dilution; Molec-
ular Probes, Eugene, OR) were used as secondary antibodies along with
the nuclear stain TO-PRO-3 (1:5000 dilution, Molecular Probes). Control
preparations consisted of tissue slices that lacked both primary and sec-
ondary antibodies and were stained only with TO-PRO-3 and tissues
stained with secondary antibodies but lacked primary antibodies. Immu-
noreactivity was visualized with a Nikon C1 Si laser confocal imaging
system equipped with a 90i Nikon microscope (Nikon, Melville, NY).
Statistical Analysis
Intragroup differences in osteogenic differentiation were calculated
for each cell line (ie, control vs osteogenic) using a Wilcoxon signed rank
test and intercomparison differences in osteogenic differentiation among
all cell lines with the Kruskal-Wallis test and the Tukey post hoc multiple
comparison test. For each cell line, control groups served as a reference
and were set to 100%. Data were summarized as percent change in osteo-
genesis quantification relative to controls. Statistical analysis was per-
formed with Graph Pad Prism 5.0 (Graph Pad, La Jolla, CA) at a = 0.05.
Results
SCAPs isolated from a CS of apical papilla surrounded by inflamed
periapical tissues (SCAP CS) coexpressed CD73 and CD90 while being
Survival of the Apical Papilla 3
Regenerative Endodontics
negative for CD45 in >99% of all cells. Of these cells, 96.6% also ex-
pressed the MSC marker CD105 (Fig. 2A). MSC marker expression in
SCAP CS was similar to the coexpression of the same markers in the
SCAP-RP89 cell line (ie, CD73, CD90, and CD105 coexpressed in
97.8% of the cell population) (18). IPAPCs were found negative for
the marker CD45 in >99% of the cells. Of the CD45-negative cells,
91.8% coexpressed CD73 and CD90, and of these cells, 66.3% also
expressed the marker CD105 (Fig. 2B).

Figure 2. Scatter plots showing the expression of MSC markers in the SCAP CS and IPAPC cell lines by multicolor flow cytometry analysis. After doublet discrim-
ination, CD45-negative cells were analyzed for CD73, CD90, and CD105 using gates based on the unstained control. (A) The SCAP CS. Plots a, b, and c show single
expression of CD73, CD90, and CD105, respectively, and plot d shows coexpression of all 3 markers (96.6%). (B) IPAPCs. Plots e, f, and g show single expression
of CD73, CD90, and CD105, respectively, and plot h shows coexpression of all 3 markers for this cell line (66.3%). SSC, side scatter.

4 Chrepa et al. JOE — Volume -, Number -, - 2016


Figure 3. Quantitative osteogenesis of the cell line SCAP CS, SCAP-RP89, and IPAPCs. Cells were cultured for 3 weeks in either control or osteogenic medium. (A)
Alizarin red staining shows the robust mineralization potential of all cell lines compared with their respective control. (B) Quantification of osteogenesis confirms
the significantly greater mineralization of all cell lines compared with their control (all P values <.05). SCAP CS expressed significantly greater mineralization
compared with both SCAP-RP89 and IPAPCs (P < .001). *P < .05. **P < .01. ***P < .001. n.s: not significant.
All 3 cell lines showed a robust mineralization potential when
induced in osteogenic media compared with their controls (Fig. 3A
and B). SCAP CS showed a more than 3-fold increase in mineralization
relative to the control group (P < .001), and it was significantly greater
than the mineralization found in the SCAP-RP89 and IPAPCs groups
(P < .001). There was no significant difference in mineralization be-
tween the SCAP-RP89 cell line and the IPAPC group (Fig. 3B).
Confocal microscopic examination of the sections from apical
papilla CS, apical papilla (normal pulp), coronal pulp (normal
pulp), and inflamed periapical tissue specimens showed specific stain-
ing for CD105 and vWF in all specimens (Fig. 4A–D). CD105 was
colocalized with the endothelial factor vWF in vascular structures in
apical papilla (normal pulp), coronal pulp, and periapical lesions
(Fig. 4B–D). In apical papilla CS, CD105 was also colocalized with
vWF; however, VWF expression was distributed throughout the tissue
specimen outside the vascular compartment (Fig. 4A). The distribution
pattern of vWF in the apical papilla CS was shown in all stained slides,
and control preparations confirmed that the staining was specific. Cells
in the apical papilla (normal pulp) and apical papilla CS expressed the
MSC marker CD24 prominently, whereas prominent CD24 staining was
not evident in coronal pulp or periapical lesion specimens (Fig. 4E–H).
Expression of the inflammatory markers CD45 and CD68 was promi-
nent in the periapical lesion specimen, moderate in the apical papilla
CS, and mostly absent in the apical papilla (normal pulp) and coronal
pulp (normal pulp) specimens (Fig. 4I–L).
Discussion
In immature teeth, the apical papilla represents an enriched pool
of stem cells that play a significant role in root development and are
likely to participate in regenerative procedures (1, 7, 17). Studies
have thoroughly characterized the human apical papilla and its
residing stem cells under normal conditions (17, 18). Nevertheless,
REPs aim to achieve dental pulp regeneration in root canals under
conditions of pulp necrosis, infection, and periapical inflammation.
These conditions may alter the characteristics and phenotype of
apical papilla and SCAPs, which may affect, among other factors, the
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Regenerative Endodontics
regeneration outcome. Studies have evaluated the effect of various
stress microenvironments on SCAPs including hypoxia, serum
deprivation, and inflammation; yet, these conditions were induced in
culture (19, 20, 23). More importantly, the survival of the apical
papilla after pulpal necrosis and apical periodontitis has never been
reported. Here, for the first time, we characterized a human apical
papilla sample that was isolated from an immature tooth with pulp
necrosis and apical periodontitis. Data from this report provide
evidence that apical papilla tissues are capable of surviving pulpal
necrosis despite moderate inflammatory cell infiltration and that
endodontic infection and inflammation may alter the differentiation
potential of SCAPs in humans.
In this study, SCAPs were isolated from the clinical sample of in-
flamed apical papilla (SCAP CS) and processed for flow cytometry to
evaluate MSC marker expression. Flow cytometry analysis showed a
96.6% coexpression of the MSC markers CD73, CD90, and CD105.
These data were very similar to the MSC expression of SCAPs under
normal conditions (18), which implies that SCAPs very likely retain their
viability and stemness even in an inflammatory environment. Notably, a
lower percentage of CD73- and CD90-positive IPAPCs (66.3%) coex-
pressed the marker CD105. Because a significant influx of cells express-
ing CD73 and CD105 is introduced in the root canals during REPs (5), it
is possible that SCAPs participate more actively in this process than
IPAPCs, at least in immature teeth.
Osteogenesis quantification was further performed to assess the
differentiation potential of the SCAP CS compared with a characterized
SCAP cell line (SCAP-RP89) and IPAPCs. Notably, the SCAP CS expressed
a significantly higher differentiation potential than SCAP-RP89 and
IPAPCs. These data agree with a previous report that short-term expo-
sure of SCAPs to proinflammatory cytokines induced greater cell prolif-
eration and mineralization potential than the control group (19).
Nevertheless, they also reported that longer exposure to inflammation
had the opposite outcomes (19). In another study, histopathological
analysis of apical papilla of rat immature teeth during inflammation re-
vealed that apical papilla survived pulp infection and periapical inflam-
mation for at least 90 days while being moderately inflamed (21).
Confocal microscopic analysis in this study showed that the apical
Survival of the Apical Papilla 5
Regenerative Endodontics

Figure 4. Immunohistochemistry and confocal microscopy showing the expression of the MSC markers CD24 and CD105 and the inflammatory markers CD45/
CD68 along with the endothelial vWF) in the tissues. (A, E, and I) The apical papilla CS (ie, inflamed), (B, F, and J) normal apical papilla, (C, G, and K) normal
coronal pulp, and (D, H, and L) periapical lesion. (A–D) Arrows show the coexpression of CD105 (red) and vWF (green) in all samples. vWF appears localized in
the vascular structures in all tissues except for the (A) apical papilla CS where it appears distributed throughout the tissue specimen outside the vascular compart-
ment. (E–H) CD24 (red) was prominently expressed in the (E and F) apical papilla samples, but there was no expression noted in the (G and H) coronal pulp or
periapical tissue sections. (I–L) CD45/68 expression (red) was moderate in the (I) apical papilla CS, prominent in the (L) periapical lesion, and mostly absent in
the (J and K) normal pulp tissue samples. Nuclei are identified by TO-PRO-3 staining (blue).

papilla CS expressed markers for immune cells (CD45 and CD68 immu-
nofluorescence), showing an inflammatory infiltrate into the apical
papilla. However, the expression of these immune markers was signif-
icantly less prominent than that seen in periapical lesions. Although the
patient reported discomfort with the tooth for approximately 1 year, it
was not possible to determine for how long the apical papilla had been
exposed to inflammatory conditions before treatment. This represents a
limitation of this study. Further research evaluating the effects of factors
such as the exposure time of human apical papilla and stem cells to
endodontic infection and its subsequent inflammatory response is
warranted.
In addition to assessing inflammatory markers, confocal micro-
scopic analysis included the MSC markers CD24 and CD105 combined
with the endothelial marker vWF. This analysis assessed apical papilla
and coronal pulp specimens from normal teeth and periapical lesion
specimens along with the inflamed apical papilla (apical papilla CS)
in order to assess differences in the immunophenotype among these
tissues. The expression of CD105 in the inflamed apical papilla sample
as well as in normal apical papilla confirmed the flow cytometry results
that SCAPs maintain MSC marker expression under inflammatory con-
ditions. Studies have suggested the possibility of MSC marker CD24 to
be a specific marker for SCAP because it is highly expressed in SCAP
populations and not detected in other MSC lines (2, 23). Results
from our immunohistochemical analysis confirmed prominent CD24
expression exclusively in the apical papilla CS and normal apical
papilla that was lacking in the coronal pulp and inflamed periapical
tissues. Moreover, inflammatory conditions in the apical papilla CS
seemed to not affect the expression of this MSC marker. Another
important finding from this analysis is the robust difference in the
distribution pattern of the endothelial factor vWF in inflamed
apical papilla compared with all other samples. The vWF
immunofluorescent signal was widespread throughout the apical
papilla CS rather than compartmentalized within vascular structures
as seen in all other tissue specimens. A possible explanation of this
finding is that certain SCAP populations may have been subjected to
endothelial transdifferentiation under the inflammatory conditions
(23). This phenomenon has been extensively described for dental
MSCs as their response to tissue injury, inflammation, or other stress
microenvironments (20, 23–26). Moreover, the apical papilla has
been previously shown to be a relatively avascular tissue (27), and
the presence of endothelial cell markers throughout the apical papilla
may suggest an active process of angiogenesis. This process may be trig-
gered by the resident SCAPs and their release of angiogenic factors such
as vascular endothelial growth factor upon exposure to the hypoxic
environment of a necrotic pulp (20).
In conclusion, under inflammatory conditions, human apical
papilla was found inflamed with retained SCAP vitality and stemness
and increased osteogenic differentiation and induction of angiogenesis.

6 Chrepa et al. JOE — Volume -, Number -, - 2016


Future studies are warranted to confirm these findings and further
explore the mechanisms of these environment-mediated changes on
human apical papilla and SCAPs as well as their effect on dental pulp
regeneration outcome.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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