Clinical Research
Evaluation of the Delivery of Mesenchymal Stem Cells into
the Root Canal Space of Necrotic Immature Teeth after
Clinical Regenerative Endodontic Procedure
Tyler W. Lovelace, DDS,* Michael A. Henry, DDS, PhD,* Kenneth M. Hargreaves, DDS, PhD,*†
and Anibal Diogenes, DDS, MS, PhD*
Abstract
Introduction: Immature teeth with open apices treated
with conventional nonsurgical root canal treatment
often have a poor prognosis as a result of the increased
risk of fracture and susceptibility to recontamination.
Regenerative endodontics represents a new treatment
modality that focuses on reestablishment of pulp vitality
and continued root development. This clinical procedure
relies on the intracanal delivery of a blood clot (scaffold),
growth factors (possibly from platelets and dentin), and
stem cells. However, to date, the clinical presence of
stem cells in the canal space after this procedure has
not been demonstrated. The purpose of this clinical
study was to evaluate whether regenerative endodontic
procedures are able to deliver stem cells into the canal
space of immature teeth in young patients and to iden-
tify the possible tissue origin for these cells. Methods:
After informed consent, the first appointment consisted
of NaOCl irrigation and treatment with a triple antibiotic
paste. One month later, the root canal space was irri-
gated with sterile saline, and bleeding was evoked
with collection of samples on paper points. Real-time
reverse-transcription polymerase chain reaction and
immunocytochemistry were conducted to compare the
gene transcripts and proteins found in the root canal
sample with levels found in the systemic circulation.
Results: Molecular analyses of blood collected from
the canal system indicated the significant accumulation
of transcripts for the stem cell markers CD73 and
CD105 (up to 600-fold), compared with levels found in
the systemic blood. Furthermore, this effect was selective
because there was no change in expression of the differ-
entiation markers ALK-P, DSPP, ZBTB16, and CD14.
Histologic analyses demonstrated that the delivered cells
expressed both CD105 and STRO-1, markers for a subpop-
ulation of mesenchymal stem cells. Conclusions: Collec-
tively, these findings demonstrate that the evoked-
bleeding step in regenerative procedures triggers the
From the *Department of Endodontics and †Departments of Physiology, Pharmacology, and Surgery, University of Texas Health Science Center at San Antonio, San
Antonio, Texas.
Address requests for reprints to Anibal Diogenes, DDS, MS, PhD, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX
78229-3900. E-mail address: Diogenes@uthscsa.edu
0099-2399/$ - see front matter
Copyright ª 2011 American Association of Endodontists.
doi:10.1016/j.joen.2010.10.009
JOE — Volume 37, Number 2, February 2011
significant accumulation of undifferentiated stem cells into the canal space where these
cells might contribute to the regeneration of pulpal tissues seen after antibiotic paste
therapy of the immature tooth with pulpal necrosis. (J Endod 2011;37:133–138)
Key Words
Confocal, dental pulp, genotype, immature necrotic teeth, mesenchymal stem cells,
regenerative endodontics, revascularization, RT-PCR, SCAP, stem cell therapy.
E ndodontic therapy in permanent teeth diagnosed with pulpal necrosis and immature
root development is fraught with challenges. The primary objective of endodontic
therapy in infected root canal systems is to disinfect the root canal system through chem-
ical and mechanical means (1). However, in teeth with immature root development, the
removal of microorganisms by mechanical means is limited as a result of the thin, fragile
dentinal walls of the roots. Disinfection of the root canal system in these cases often
relies on irrigation and intracanal medicaments. After completion of traditional
endodontic therapy, these teeth often have a poor crown-to-root ratio and are suscep-
tible to fracture (2, 3). Recently, an alternative, biologically based, regenerative
approach has been advocated on the basis of prior revascularization studies from
the trauma literature (4–7). The publication of several case series has led to
growing recognition of the potential for successful outcomes by using regenerative
procedures in treating the necrotic immature permanent tooth (8–11).
Multiple studies showed continued root development can be accomplished after
disinfection of the root canal system, evoked bleeding inside the root canal, and
adequate coronal seal (8, 10, 12–17). The use of these treatment protocols can
result in radiographic and clinical evidence of healing and subsequent root
development that has been attributed to a regeneration of tissue. The exact
mechanism and source of the cells responsible for the radiographic evidence of
continued root development remain unknown. However, the identification of an
enriched population of mesenchymal stem cells within the apical papillae (SCAP) of
immature teeth has led to the suggestion that these cells might contribute to the
regenerative response that follows these clinical procedures (18). Although regenera-
tive protocols include a step that induces bleeding within the canal space after the
manipulation of periapical tissues, it is not known whether this step triggers the release
of stem cells into the root canal space. This issue represents an important gap in knowl-
edge because tissue engineering involves the controlled delivery of stem/progenitor
cells as well as a scaffold and growth factors (18), so knowledge regarding the potential
source of stem cells is critical to the further development of pulpal regenerative
Delivery of Mesenchymal Stem Cells by Regenerative Endodontic Procedure 133
Clinical Research
procedures. Some have even questioned the possible role of stem cells
in these regenerative responses, and so the possible presence of stem
cells within the root canal space after a regenerative procedure needs
to be evaluated. Accordingly, in the present study we evaluated the
delivery of mesenchymal stem cells into root canal systems of immature
necrotic permanent teeth in human subjects.
Materials and Methods
Patient Recruitment
The Institutional Review Board of the University of Texas Health
Science Center at San Antonio approved this study, and the informed
consent of all human subjects and their guardians was obtained. Inclu-
sion criteria consisted of patients between 7 and 16 years of age with an
immature permanent maxillary or mandibular single-rooted immature
tooth with open apices (>2.5 mm of apical radiographic diameter) with
a diagnosis of pulp necrosis with apical periodontitis with lesion or
suppurative apical periodontitis and no systemic health problems that
might interfere with healing of apical periodontitis (ie, immunocom-
promised, long-term steroid usage, etc). A total of 12 patients were
included in this study and consisted of 6 boys and 6 girls with an average
age of 10.5  2.7 years. Twelve teeth (1 per patient) were included in
the study and were composed of 7 maxillary central incisors with history
of complicated crown fracture and 5 mandibular premolars with history
of dens evaginatus. All teeth were diagnosed with necrotic pulp and
symptomatic apical periodontitis with radiographic evidence of a perira-
dicular lesion.
Regenerative Procedure and Sample Collection
The regenerative protocol consisted of the following procedures.
Patients were anesthetized by using 1.8–3.6 mL of 3% mepivacaine
without vasoconstrictor, with a buccal infiltration for maxillary teeth
or an inferior alveolar nerve block for mandibular teeth. Subsequently,
teeth were isolated with a rubber dam; caries, if present, was removed;
and endodontic access was performed. The working length was deter-
mined radiographically by inserting #15 K-file size into the canal at the
estimated preoperative canal length. The canals were disinfected with
passive positive pressure irrigation with 20 mL of 6% NaOCl and 10
mL saline via a Maxiprobe (Dentsply Rinn, Elgin, IL) needle placed 1
mm from the radiographic apex of the tooth. The canals were then dried
with paper points. The triple antibiotic paste consisted of USP grade
antibiotic powders compounded in a 1:1:1 ratio of metronidazole, ci-
profloxacin, and minocycline by a local pharmacy (Champs Pharmacy,
San Antonio, TX) and mixed in double-distilled sterile water to form
a paste-like consistency. A Food and Drug Administration Investiga-
tional New Drug permit was obtained for the off-label use of these anti-
biotics in this study. The antibiotic paste was delivered into the root
canal spaces with the use of a Centrix syringe attached to a high-
viscosity Accudose needle tube (Centrix, Shelton CT) under high-
power magnification. The access was sealed with Cavit (3M, St Paul,
MN).
Patients returned for the second treatment visit 1 month later. A
positive blood aspirate (systemic blood sample) was collected in the
cartridges during local anesthetic injection and immediately placed
into an RNA isolation lysis buffer (RNeasy MiniKit; Qiagen, Valencia,
CA). Teeth were isolated, accessed, and irrigated with sterile 0.9%
saline; paper points were placed into the canal and allowed to absorb
the saline intracanal samples for 2 minutes; the paper points were
placed and removed under microscopic magnification to ensure that
they did not touch the canal walls or the external surface. The paper
points were immediately placed into RNA isolation lysis buffer (Qiagen).
Bleeding from the apical region was achieved by rotating a pre-curved
134 Lovelace et al.
K-file size #25 at 3–5 mm beyond the working length. After microscope
visualization of intracanal bleeding under high-power magnification,
a second paper point was placed intracanal for 2 minutes to collect
an intracanal blood sample, which was placed in RNA isolation lysis
buffer (Qiagen).
RNA Isolation and Real-time Reverse-Transcription
Polymerase Chain Reaction
Total RNA was isolated by using the RNeasy Mini Kit, and any poten-
tial genomic DNA contamination was removed by DNAse treatment
(DNA-free kit; Ambion, Austin, TX). Only RNA samples with an optical
density A260/A280 ratio between 1.8 and 2.1 (indicative of RNA quality)
were used (19). Therefore, if 1 or more RNA samples from a patient did
not meet this prerequisite, the patient was excluded from the study. Of
the total 12 sets of samples collected from 12 separate regenerative
procedures, only 8 met this quality control inclusion criterion. The
excluded samples were from 3 maxillary incisors with history of compli-
cated crown fracture and from 1 mandibular premolar with history of
dens evaginatus. Total RNA isolated from the included samples was used
as template in 2-step real-time reverse-transcription polymerase chain
reaction (RT-PCR) reactions. Initially, RNA templates (0.5 mg) were
used in complementary DNA (cDNA) synthesis reactions by using
Superscript reverse transcriptase II kit (Invitrogen, Carlsbad, CA).
The cDNA templates were then used in real-time PCR reaction by using
TaqMan Assays on Demand (Applied Biosystems, Foster City, CA) con-
sisting of previously validated primers and probes specific for the
mesenchymal stem cell markers: cluster of differentiation 105
(CD105) (aka endoglin gene assay #Hs00923997 g1) (20), cluster
of differentiation 73 (CD73) (aka NT5E gene assay #Hs00159686
g1) (20), zinc finger and BTB domain containing protein 16
(ZBTB16) (assay #Hs00232313 m1), and cyclin D2 (assay
#Hs00922419). In addition, the immune cell marker CD14 (assay
#Hs02621496 s1), the dentinogenic/osteogenic marker dentin sialo-
phosphoprotein (DSPP) (assay #Hs00171962) (21), the osteogenic
marker alkaline phosphatase (ALK) (assay #Hs03046558 s1) (22),
and an internal loading control (housekeeping gene) 18S (ribosomic
18S subunit gene; assay #Hs03003631 g1) were used. The reactions
were run in triplicates of 25 mL, and cycle threshold (Ct) values ob-
tained after the reactions were stopped at 45 amplification cycles.
The Ct values for each target gene were normalized to the endogenous
control (18S) values. The comparative delta-delta Ct method was used
to normalize the data on the basis of the endogenous reference and to
express it as the relative fold change in relation to the values obtained
from the patient’s own systemic blood control levels after the exclusion
criteria were verified by comparing primer efficiencies (23, 24).
Immunocytochemistry and Laser Confocal Microscopy
In addition to RNA isolation and real-time RT-PCR reactions,
samples were also evaluated by immunocytochemistry. Intracanal blood
samples were taken during treatment, and paper points were immediately
placed in cold phosphate-buffered saline (PBS) buffer. The PBS-
containing cells were pipetted onto glass slides and allowed to air dry.
Samples were then fixed by submersion in 100% ETOH for 10 minutes,
permeabilized, and blocked for nonspecific protein binding sites with
blocking solution consisting of 4% normal goat serum (Sigma, St Louis,
MO), 2% bovine gamma-globulin (Sigma), and 0.3% Triton X-100
(Fisher Scientific, Pittsburgh, PA) in PBS for 60 minutes before the incu-
bation of primary antibodies consisting of an anti-CD105 (Endoglin,
M3527; Dako, Carpinteria, CA;1:500 dilution) raised in rabbit, anti-
STRO-1 (MAB10; R&D Systems, Minneapolis, MN; 1:100 dilution) raised
in mouse, or anti-CD73 (SC-101344; Santa Cruz Biotechnologies, Santa
JOE — Volume 37, Number 2, February 2011

Cruz, CA; 1:200 dilution) for approximately 12 hours at room tempera-
ture. Slides were rinsed in PBS and then incubated in secondary anti-
mouse immunoglobulin G conjugated to Alexa-488 fluorophore (Molec-
ular Probes, Eugene, OR) to detect each primary antibody’s immunore-
activity (Molecular Probes). All slides were also incubated with the
nuclear stain TO-PRO-3 (Molecular Probes; 1:5000 dilution). Primary
and secondary antibodies were both diluted in blocking solution. Slides
were rinsed in PBS, water, air-dried, and coverslipped with Vectashield
(Vector Labs, Burlingame, CA), and immunoreactivity was visualized
with a Nikon C1 si laser confocal imaging system equipped with 90i Nikon
microscope (Nikon, Melville, NY).
Statistics
Gene expression data were analyzed with one-way analysis of vari-
ance with Bonferroni post hoc test, and significance was set at P <.05 by
using the Graph Pad Prism 5.0 version software (Graph Pad, La Jolla, CA).
Results
Eight patients met the inclusion criteria and consisted of 5 boys
and 3 girls with an average age of 9.3  2.1 years. All teeth included
were diagnosed as having pulpal necrosis and symptomatic apical pe-
riodontitis and consisted of 4 maxillary central incisors and 4 mandib-
ular premolars. Subset data analysis revealed no statistically significant
effect of tooth type on the expression of the genes evaluated. Collectively,
in comparison to levels of mesenchymal stem cell markers observed in
the systemic blood, the triggered bleeding into the root canal system
evoked a 178.9  90.3 fold increase in CD73 (P < .05) and a 604.4
 262.8 increase in CD105 (P < .05) transcripts (Fig. 1A). Indeed,
the systemic and intracanal saline samples demonstrated virtually no
detectable expression of CD73. The expression of CD105 was increased
26.13  4 fold (P < .05) in the intracanal saline sample when
compared with the expression in systemic blood (Fig. 1A). Thus, the
manipulation of periapical tissues and evoked-bleeding step that is
a common step used in different regenerative protocols triggers
a substantial influx of cells containing mRNA transcripts encoding
mesenchymal stem cell marker genes into the root canal system.
We next evaluated whether the bleeding step triggers the influx of
differentiated cells. The alkaline phosphatase gene (ALK-P) was not de-
tected in any of the samples. However, the expression of DSPP was de-
tected in 25% (2 of 8) of the intracanal blood samples, but its overall
mean fold expression change (35.56  25.3) was not statistically
different when compared with the DSSP expression seen in systemic
blood (Fig. 1B). The immune cell marker CD14 was detected in 25%
(2 of 8) of the saline samples (106.6  104.2 fold change; not statis-
tically significant) and 50% (4 of 8) of the intracanal blood samples
(14.39  6.1 fold change; P < .05) (Fig. 1C). The ZBTB16 gene
was detected in 50% (4 of 8) of the intracanal blood samples, but
the overall expression change was not statistically significant when
compared with the systemic blood levels. However, a significant up-
regulation in the expression of the cyclin D2 gene was detected in
50% (4 of 8) of the saline samples (14.39  6.1 fold change; P <
.05), whereas no difference was detected in the expression of cyclin
D2 in intracanal blood samples when compared with the systemic blood
samples (Fig. 1D).
We next used immunocytochemistry to characterize the expres-
sion of stem cell markers within cellular profiles found in blood smears
collected from the patient’s root canal system. The mesenchymal stem
cell markers CD105, CD73, and STRO-1 were detected in cells collected
from the canal space after the evoked-bleeding step in the confocal
micrographs (Fig. 2A, B, and C, respectively).
JOE — Volume 37, Number 2, February 2011
Clinical Research
Discussion
The involvement of stem cells in regenerative procedures has been
hypothesized, but to the best of our knowledge, it has never been clin-
ically demonstrated in patients. In the present study, we have demon-
strated with real-time RT-PCR and immunocytochemistry that the
evoked-bleeding step of a widely used regenerative endodontic treat-
ment protocol (13) results in the delivery of stem cells into root canal
systems and that the levels of their molecular markers are up to several
hundred fold greater than levels observed in the systemic blood
collected from the same patients. The evoked-bleeding step involves
the manipulation of the periapical tissues, and it is this manipulation
step that appears to release stem cells from these tissues and results
in their delivery to the canal system. Even though the exact source of
these stem cells has yet to be determined, one likely source is from
the apical papilla because it contains an enriched population of mesen-
chymal stem cells. However, these cells might be derived from several
sources, including systemic blood or local tissues such as bone, peri-
odontal ligament, dental follicle, dental pulp, or the apical papilla
(25–29). The data presented here not only demonstrate the delivery
of stem cell markers into the root canal space, but they indicate that
these cells most likely originate from local tissues adjacent to the
apex of the root and not from the systemic circulation.
Mesenchymal stem cells, as stated by the Society of Cellular
Therapy, must express the cluster of differentiation markers CD105,
CD90, and CD73 in a population of cells (20). In addition, these
markers have been used to isolate and clonally expand enriched mesen-
chymal stem cell cultures (30). Thus, we evaluated the expression of the
CD105 and CD73 genes as a dependent measure of the relative presence
of stem cells. We found that the expression of both CD105 and CD73 was
substantially and significantly increased after the evoked bleeding from
the periradicular tissues from immature teeth with open apices. These
results are consistent with the hypothesis that the mechanical disruption
of periradicular structures such as the apical papillae releases a substan-
tial quantity of stem cells from their niche. Thus, the blood invading the
canal space after the evoked-bleeding step transports these cells to the
target area (pulp canal space). It has been demonstrated that mesen-
chymal stem cells, probably bone marrow stem cells, can be found in
the circulatory blood (31). Therefore, we also collected systemic circu-
latory blood samples and normalized all the data to the expression level
of each marker to levels observed in the patient’s systemic blood
sample. Thus, the increased expression of mesenchymal stem cell
markers is not due to the delivery of normally circulating stem cells,
but instead it strongly implicates the local delivery of stem cells from
the periradicular tissues into the root canal space. Interestingly, the
expression or detection of the mesenchymal stem cell marker CD105
in samples collected during the saline flush before evoked bleeding
suggests that a few stem cells were already detached or became
detached from their niche after a gentle saline irrigation of the canal.
It is possible that these cells could be surviving dental pulp stem cells
from the apical pulp tissue that retained vitality despite advanced apical
periodontitis, or these cells could have originated from the apical
papillae or other periradicular structures such as periodontal ligament
and bone.
The antibody STRO-1 was first characterized as a monoclonal anti-
body that recognizes a subpopulation of mesenchymal stem cells
described as colony-forming units-fibroblasts because mesenchymal
stem cells often resemble the morphology of fibroblasts in vitro (32,
33). Although STRO-1 has been widely used as a true mesenchymal
stem cell marker, the gene that encodes this protein is unknown.
Thus, only the protein expression of STRO-1, and not its corresponding
mRNA transcript, could be evaluated in this study. Nonetheless, we have
Delivery of Mesenchymal Stem Cells by Regenerative Endodontic Procedure 135
Clinical Research

Figure 1. Evoked-bleeding step in endodontic regenerative procedures in immature teeth with open apices leads to significant increase in expression of undif-
ferentiated mesenchymal stem cell markers in the root canal space. Systemic blood, saline irrigation, and intracanal blood samples were collected during second
visit of regenerative procedures. Real-time RT-PCR was performed by using RNA isolated from each sample as template, with validated specific primers for target
genes and 18S ribosomal RNA endogenous control. (A) Expression of mesenchymal stem cell markers CD73 and CD105 was up-regulated after the evoked-bleeding
step in regenerative procedures. (B) Expression of osteogenic marker ALK-P and dentinogenic marker DSPP was unaltered after the evoked-bleeding step in regen-
erative procedures. (C) Expression of innate immune response cell marker (CD14) was significantly detected at the second visit (saline sample) and after intracanal
bleeding. (D) Differentiation marker ZBTB16 was not significantly changed during the regenerative procedure, whereas the expression of the proliferation marker
cyclin D2 was significantly up-regulated in the saline irrigation sample before the evoked-bleeding step. Real-time RT-PCR was performed by using sample template.
Data were normalized to the housekeeping gene 18S levels and presented as mean  standard deviation fold increase in relation to systemic blood levels for each
gene and analyzed with one-way analysis of variance with Bonferroni post hoc test (n = 8; *P < .05; **P < .01; n.s., not statistically significant).

used a well-characterized antibody against STRO-1 in addition to anti-
bodies against CD105 to demonstrate, with the use of immunocyto-
chemistry, that cells delivered after the evoked bleeding in
regenerative procedures expressed the required CD105, CD73, and
STRO-1 markers of mesenchymal stem cells. Also, immunohistochem-
ical evaluation of these blood-delivered cells revealed that most cells
had intact nuclei (visualized by TO-PRO-3 staining) and normal
morphology that differed from other types of cells. Collectively, these
findings suggest that cells positive for CD105, CD73, or STRO-1 were
viable stem cells found in root canals after regenerative endodontic
procedures.
The ALK-P and DSPP genes are known to be expressed in cells of
osteoblastic and odontogenic cell lineages, respectively (34). Although
DSPP is constitutively expressed in mature odontoblasts, its expression
is significantly increased during the odontoblastic differentiation
process (21). In the present study, the expression of these 2 genes
was not significantly increased after regenerative procedures in imma-
ture teeth (only DSPP was detected in 2 of 8 samples). This finding
suggests that the stem cells detected after regenerative procedures
were undifferentiated cells and had not yet been committed to either
osteoblastic or odontoblastic lineages.
The transcription factor ZBTB16 (aka promyelotic leukemia zinc
finger transcription factor) gene is up-regulated during osteoblastic
differentiation of stem cells because it is a transcription activator of
the runx2, collagen 1A, and alkaline phosphatase genes (35). In the
present study, its expression was not detected in the immature teeth.
This finding provides an additional line of independent evidence that
the stem cells from the immature teeth were undifferentiated.

136 Lovelace et al. JOE — Volume 37, Number 2, February 2011

 Clinical Research

Figure 2. Mesenchymal stem cells were delivered into root canal spaces during regenerative procedures in immature teeth with open apices. Cells collected from
intracanal blood samples after the evoked-bleeding step or from systemic blood were stained with antibodies against CD105, CD73, or STRO-1 and evaluated with
a laser-scanning confocal microscope. (A) Cells in intracanal blood samples collected after evoked-bleeding step showed expression of mesenchymal stem cell
marker and CD105 (green in A), CD 73 (green in B), and STRO-1 (green in C), whereas nuclei appear blue as stained with TO-PRO-3.

Cyclin D2 is a gene involved in cell cycle regulation, and its expres-
sion is detected and increased when stem cells leave the arrested stage
and begin to proliferate during the Go/S cell-cycle transition (36, 37).
We detected cyclin D2 in the saline samples of immature teeth (3 of 8
samples) but failed to detect a significant increase of cyclin D2 in the
intracanal blood samples. It is possible that there were surviving stem
cells already present in the root canal space undergoing proliferation
before the evoked-bleeding step. Interestingly, the expression was not
significantly increased in the samples after the intracanal-bleeding
step. This might be due to the finding that stem cells are at an inhibited
proliferation stage (arrested at Go) when in their niche (38, 39).
Because samples were collected immediately after these cells were
released from the manipulation of periapical tissues that might
include the apical papillae (stem cell niches), we believe that
transcription of the cyclin D2 gene had not yet been initiated.
CD14 is a marker for the innate response immune cells. It is highly
expressed in macrophages, dendritic cells, and to a lesser extent in
neutrophils (40). In this study, we detected a significant expression
of CD14 in both saline (2 of 8) and intracanal bleeding (4 of 8) samples
in immature teeth. However, because of the variability of the findings,
only the intracanal bleeding sample was found to be statistically signif-
icant. This result is consistent with the prominent presence of innate
response immune cells in apical periodontitis (41, 42). At the
second visit after disinfection and triple antibiotic mix treatment, all
patients had complete resolutions of symptoms (ie, no pain, sinus
tracts, or swelling); therefore, the detection of the CD14 marker at
the second visit is indicative of residual presence of neutrophils and
macrophages.
One limitation of the present study is the inability to follow the cells
over time. The real-time RT-PCR detection of these markers is merely
a snapshot of the cells in time, and it represents the collective gene
expression profile of the cells. The expression of genes and proteins
in individual cells from this population of cells warrants more research.
In addition, there is a myriad of genes involved in stem cell proliferation
and differentiation that were not investigated in this study. One of the
technical difficulties of this study is related to the very limited quantity
of RNA sample collected in paper points. Also, only RNA samples that
were of high quality with an optical density A260/A280 ratio between
1.8 and 2.1 (high quality RNA samples) were used. In the present study,
12 regenerative procedures were performed, but only samples from 8
regenerative procedures met this inclusion criterion and were included
in the real-time RT-PCR experiments. Although we detected statistically
significant differences in the expression of some of the genes, studies
with larger sample sizes, perhaps multicenter studies, are warranted.
In conclusion, we have demonstrated for the first time that mesen-
chymal stem cells are delivered into root canal spaces during regener-
ative endodontic procedures in immature teeth with open apices. We
believe that these findings provide the biological foundation for the
involvement of stem cells in the continued root development and regen-
erative response that follow this clinically performed procedure.
Further research is needed to evaluate how to increase the proliferative
and differentiation capacity of these cells in the target tissues, the root
canal space and the periradicular environment. It is likely that the
regeneration of pulpal tissues can be made even more predictable by
the application of tissue engineering principles to use scaffolds, growth
factors, and stem/progenitor cells to develop optimal and reliable
endodontic regenerative procedures.
Acknowledgments
This research was supported in part by a grant from the Amer-
ican Association of Endodontists Foundation.
The authors deny any conflicts of interest related to this study.

JOE — Volume 37, Number 2, February 2011 Delivery of Mesenchymal Stem Cells by Regenerative Endodontic Procedure 137
Clinical Research
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JOE — Volume 37, Number 2, February 2011