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Pulp RegenerationTranslational Opportunities

Healing after Regenerative Procedures

with and without Pulpal Infection
Ashraf F. Fouad, DDS, MS, and Prashant Verma, DDS
Regenerative endodontic procedures for immature teeth
with pulp necrosis have gained a lot of interest. Basic
scientific research has documented the potential for
dental pulp regeneration in preclinical studies. A number
of case reports and case series have shown the control
of clinical infection and increases in thickness and length
of the roots. Preoperative infection is an important
factor that predicts outcome in nonsurgical endodontic
treatment in mature teeth and seems to also be an
important determinant of outcome in treating immature
teeth. However, antimicrobial strategies for the immature tooth in which pulp regeneration is contemplated
are different from those used in mature cases. This is
because of the interest in disinfecting the root canal to
a higher level to promote tissue growth without disrupting the bioactive potential of root dentin and the
viability of stem cells from which the regenerated tissue
would develop. This review addresses the factors
involved in making clinical decisions in this area in light
of the information available on the microbiology of
endodontic infections, the efficacy to antimicrobial
strategies, and the outcomes of regenerative and
alternative procedures. (J Endod 2014;40:S58S64)

Key Words
Endodontics, irrigants, molecular microbiology, pulp

From the Department of Endodontics, Prosthodontics and

Operative Dentistry, School of Dentistry, University of Maryland, Baltimore, Maryland.
This paper is based on a presentation from the International
Association for Dental Research (IADR) Pulp Biology and
Regeneration Group Satellite Meeting, which was held March
2426, 2013 in San Francisco, California.
Address requests for reprints to Dr Ashraf F. Fouad, Department of Endodontics, Prosthodontics and Operative Dentistry,
School of Dentistry, 650 West Baltimore Street, Baltimore,
MD 21201. E-mail address:
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.

ental pulp regeneration has received considerable interest in recent years.

Clearly, the main impetus for this interest is the clinical problem of pulp
necrosis with established infection before complete maturation of the tooth root.
Although this is a formidable problem, it is generally accepted that cases with
this presentation constitute a small proportion of all cases with pulpal pathosis.
However, it is also generally recognized that if predictable treatment procedures
can be achieved to control the infection and promote pulp regeneration in
cases with immature teeth, these procedures could potentially be extended to the
treatment of the vast majority of endodontic infections that occur in mature teeth,
as was recently proposed (1, 2).
Pulp regeneration in an immature tooth that was rendered pulpless by trauma,
caries, or a congenital abnormality is advantageous because of the potential for
strengthening of the tooth to the same extent as mature teeth. In addition, the presence
of vital pulp in any tooth is preferable biologically compared with a filling material
because it would restore the host immune response to the pulp and could also
potentially regenerate any missing coronal dentin. In this article, the factors that
determine success in promoting tooth maturation, the central role that pulp infection
and its elimination play, and the different antimicrobial strategies for controlling
infection are reviewed.

Pulp Revascularization without an Established Infection

Pulp revascularization procedures have been advocated in immature teeth
for decades. It has generally been recognized that for teeth with immature apex
after traumatic avulsion with short extraoral time, intrusive or extrusive luxations,
replantation/repositioning of teeth, or any transplantation of teeth with immature
apex, the teeth are monitored for spontaneous revascularization (36). In these
clinical conditions, the prognosis for revascularization and continued root
maturation ranges from 33%100%. Moreover, cases have been described in
which revascularization is attempted by the clinician in the absence of any evidence
for infection and is also shown to yield clinical success (7, 8) and histologic
revascularization (9). The contemporary recommendations for treating traumatic
injuries of immature teeth in the absence of active infection are to promote
revascularization with minimal clinical intervention, if possible (Fig. 1AD), but
monitor the case carefully in case the pulp becomes necrotic. The general
understanding in these cases is that if there is no infection or minimal contamination
without an established bacterial biofilm, host responses will allow sufficient
connective tissue to revascularize the pulp space through the relatively large apical
foramen and continue mineralization, leading to increases in the width and length
of the root. Pulp revascularization through the immature apex or a mature apex
that had been resected was shown in principle in older animal studies (1013). In
fact, the success of revascularization after traumatic injuries of immature teeth has
been shown to increase with the size of the apical foramen (14).

Pulp Revascularization with an Established Infection

The situation is very different for a case with necrotic pulp and an established
infection manifesting clinically as pulp necrosis with an acute or chronic abscess
or a radiographically visible apical lesion. In these cases, the infection has been established for a sufficient duration to allow the development of bacterial biofilms inside
the root canal. In addition, cases in which spontaneous revascularization fails present


Fouad and Verma

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Figure 1. (A) A mandibular right first premolar (#28) was inadvertently extracted during the extraction of a deciduous tooth and then promptly replanted in
this 10-year-old patient. (B) The 6-month follow-up shows normal root maturation of #28. (C) The 1-year follow-up shows continued root development #28.
(D) A radiograph of the contralateral tooth (#21) at the same time as C shows that #28 had a substantial increase in mineralization and diminution of root canal
size compared with #21.

with some form of an infection. This may include the typical signs
mentioned before and/or a lack of continued root development or
external infection-related (inflammatory) root resorption. These cases
have historically been thought to not respond to the revascularization
procedures because of the inability to disinfect the root canal to a
degree that would permit regeneration of vital pulp (15). Thus, the
important innovation in the last decade has been the demonstration
through case reports and case series that cases with established
clinical infections could be disinfected sufficiently to allow the
revascularization process to yield what appears to be clinical success.
This disinfection has been achieved through a variety of methods but
mainly with the use of various antibiotics, antibiotic combinations, or
calcium hydroxide.
The apparent success in these cases that have been described is
primarily related to the control of the established infection and the
radiographic healing of the periapical lesion. Although this is an
important outcome, it is not the original outcome for which regenerative procedures are contemplated. Apexification procedures with
calcium hydroxide or more recently with a mineral trioxide aggregate
(MTA) plug have actually been shown to yield the same outcome.
Furthermore, there are many cohort studies for MTA apexification
now showing clinical success, primarily in controlling periapical
infections (1620).
As stated previously, the interest in regenerative procedures
seeks another more important outcome related to strengthening of
the teeth involved. This outcome is difficult to evaluate. Clinicians
can show control of the endodontic infection within 12 years;
however, the strengthening of teeth requires decades of monitoring.
Calcium hydroxide apexification has been shown to fail in many cases
within 5 years, particularly in cases with very thin dentin (21). It
is speculated that this may be related to weakening of dentin
exposed to long-term calcium hydroxide (22, 23). MTA does not
appear to weaken dentin like calcium hydroxide does (24); however,

JOE Volume 40, Number 4S, April 2014

the tooth remains structurally weak and may fracture in the long-term
(Fig. 2AH).

Transformation of the Oral Microbiome

from the Oral to the Endodontic Environment
Because the development of an endodontic infection plays such a
critical role in treatment considerations and the success of regenerative
procedures, it is essential to examine the type and characteristics of
infection that results after pulp necrosis. This allows the clinician to
understand the location of bacterial biofilm, the microbial virulence
and adhesion characteristics, and the antibiotic sensitivity of the
organisms involved. This knowledge would assist in identifying the
best antibacterial strategies.
Naturally, endodontic microbiology has gone through different
phases that parallel advances in the science of microbiology. For
most of the research performed in the 20th century, culturing of root
canal microflora was the state of the art, and clinical decisions were
frequently based on cultivation results. Culturing is still the gold
standard when it comes to the identification of antibiotic resistance
or the study of virulence traits. It is also an important determinant
that persistent microorganisms after treatment modalities are alive.
However, it has recently been determined that only about 10% of the
human microbiome (the bacteria that occupy different sites in the
human body) is cultivable (25). In addition, many of the cultivable
bacteria may be rendered temporarily uncultivable if they are
exposed to reagents or techniques that may exert metabolic stress
but resume their cultivability once these stressors are eliminated
(26, 27). Therefore, culturing information is at best incomplete in
defining the microbial content of infected root canals.
Several generations of molecular technologies have led to a
dramatic improvement in our knowledge of endodontic microbiology.
Older molecular studies merely investigated the presence of organisms

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Pulp RegenerationTranslational Opportunities

Figure 2. (A) A maxillary left central incisor (#9) was avulsed, replanted within an hour, and splinted. (B) Evidence of infection-related (inflammatory) root
resorption detected within 4 weeks. (C) Endodontic treatment was initiated, and calcium hydroxide medicament was placed. (D) After 5 weeks, the canal was
filled with MTA. (E) The 1ne-year and (F) 2-year follow-up show the success of the treatment. (G and H) The patient presented 4 years after treatment with a
periapical lesion and horizontal root fracture. The tooth had to be extracted.

that had been identified by culturing or used the inefficient and

expensive cloning and sequencing methodologies (28). More recently,
next-generation sequencing studies have revealed previously
unrecognized diversity and richness in endodontic infections (29,
30). There are also remarkable differences observed in specimens
from different patients (29, 31) and the expression of different
virulence traits in different strains of the same microbial
species (32, 33). These facts make it very difficult to identify specific
microorganisms that are responsible for clinical symptoms,
resistance to antimicrobial agents, and markers of disease
persistence. Newer strategies have sought to link the vastly diverse
endodontic infections to the normal oral microbiota of the same
individuals from which they originate (34). The basic principle here
is to identify the members of the endodontic microbiome that become
over-represented relative to the original source in acute or symptomatic
conditions and would therefore be the true putative pathogens. This
study showed remarkable diversity of the endodontic microbiome in
3 locations: normal oral cavity, necrotic root canal space, and apical
abscess (Fig. 3). It can be seen from the differential abundance of
microbiota how the proportion of streptococci and Veillonella spp.,
which are very abundant in the oral cavity, decrease in endodontic
infections and the abundance of gram-negative anaerobes such as
Fusobacterium spp., Prevotella spp., and Porphyromonas spp. and
the gram-positive Parvimonas spp. increase. Lesser known genera
like Afipia spp., Phocaiecola spp., Gemella spp., and 1 unclassified
genus also increase to different extents in necrotic root canal spaces

Fouad and Verma

and apical abscesses. These could play an important role in the overall
pathogenicity of the infection. This remarkable overview of the
dynamics of bacterial changes in endodontic infections provides a novel
insight into the pathogenesis of these infections.

The Role of Infection in Treatment Outcomes

Preoperative endodontic infection is generally determined by the
lack of response to vitality tests and the presence of a radiolucent
periapical lesion. Numerous outcome studies have identified
preoperative infection as the single most important factor in the success
of endodontic treatment of teeth with a mature apex. The presence of
infection reduces the chances for complete clinical and radiographic
healing by about 15%20% compared with cases with vital pulp
(3538). These findings appear to be consistent in many studies,
regardless of the proficiency of the operator and recent technological
advances in the field.
Furthermore, one prospective study (37) showed that the probability
of long-term healing was not only significantly lower in the presence of a
preoperative periapical lesion but also that larger periapical lesions, the
presence of a sinus tract, and preoperative symptoms or perforations
during treatment in these cases were associated with a worse prognosis.
These findings seem to suggest a dose-dependent and duration-dependent
effect of preoperative infection on endodontic treatment outcomes.
At the same time, it has been shown that effective debridement in
the apical third of root canals significantly improved healing. A recent
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and on the apical extraradicular root surfaces. Traditional chemomechanical debridement of the root canal leaves behind residual bacteria
(40), which are then entombed by obturation of the canal space. After
placement of a well-sealing coronal restoration, the environment is
conducive to healing in most cases. However, in regenerative endodontics, the lack of a filling in the canal space while the regenerated tissue
is developing may allow the residual bacteria to proliferate and
re-establish a biofilm. Therefore, the necessary level of disinfection is
likely higher than that accepted for traditional root canal therapy
because in traditional techniques merely lowering bacterial loads and
preventing bacterial access to the apex are conducive to healing.
Furthermore, the developing tissue needs sufficient time to establish
itself in the root canal, and, thus, the aseptic environment needs to
be maintained for a longer period of time than in traditional endodontic
therapy. Antimicrobial agents with higher levels of substantivity are
needed for this reason.
Several case reports and case series have shown successful results
of regenerative endodontic treatment in cases of preoperative infection,
including control of the infectious process, radiographic thickening of
canal walls, and continued root development (4146). However, there
are currently no clinical trials or outcome studies that have specifically
looked at the effect of preoperative infection on the prognosis of
regenerative endodontic treatment. More research is needed in this area.

The Selection of Optimal Antimicrobials

for Pulp Canal Disinfection

Figure 3. Relative abundance (in percent of total sequences) of 10 most

abundant bacterial genera in 3 sites from the same individual (normal oral
cavity [OS], necrotic root canal space [RC], and acute apical abscess [AS])
(N = 23 total samples from 8 patients) that yielded 231,519 sequences.
Each sample had an average of 70 bacterial genera (range 26132). (Modified
from Hsiao WW, Li KL, Liu Z, et al. Microbial transformation from normal oral
microbiota to acute endodontic infections. BMC Genomics 2012;13:345. Published by BioMed Central.)

randomized clinical trial showed that mechanically enlarging the canals

to a minimum of 3 sizes larger than the initial binding apical file led to
significantly higher success 1 year postoperatively (39). In this study,
there were also incremental increases in the success rate with larger
size preparations; although they were not statistically significant, they
were indicative of a dose response that warrants further study.
As noted previously, the effect of preoperative infection is also
very important in cases in which regenerative endodontics is attempted.
Preoperative infection may be the determinant of whether spontaneous
revascularization or regenerative procedures are to be contemplated.
Moreover, the traditional level of root canal disinfection may not be sufficient in cases in which vital tissues are to occupy the pulp space in
which a previous infection was present. Bacteria form biofilms on the
canal walls, in isthmuses and lateral canals, inside the dentinal tubules,
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It follows from the previous discussion that there should be some

selectivity in the choice of antimicrobial agents, given what is known
about the nature of endodontic infections. There are 2 additional factors
that further support this notion that specific antimicrobials need to be
identified for the treatment of teeth with immature apex. Given that
these teeth have a very thin dentinal wall, minimal mechanical
instrumentation is advocated so as not to further weaken the tooth
structure. However, it is important to note that without the frictional
force applied by a file to dentinal wall, bacterial biofilms remain intact
and are much more resistant to antimicrobial agents than if they were
rendered planktonic by this mechanical disruption. Therefore, a small
amount of filing is performed, the intent of which is not to shape the root
canal (such in mature teeth) but rather to create inroads through the
biofilm to allow maximum permeation by the antimicrobials.
The second important factor is that although maximum antimicrobial efficacy is needed to prevent bacterial irritation of the
revascularized/regenerated tissue, minimal toxicity of these antimicrobials on the soft and hard tissues surrounding this newly formed
tissue is critical. For example, it is known that 2.5%5.25% sodium
hypochlorite and 2% chlorhexidine are among the most effective
antimicrobials in nonsurgical endodontic treatment of teeth with
mature apex. However, in vitro and animal model studies have shown
that these materials at these concentrations may be toxic to stem cells of
the apical papilla (47), may prevent adhesion of stem cells to dentin
(48), and may abrogate the bioactivity of growth factors sequestered
in dentin (49). Therefore, of these agents, current clinical guidelines
advocate only the use of 1.25% sodium hypochlorite at the first clinical
The use of antibiotics becomes the obvious next choice because of
their selectivity, their relatively reduced toxicity, and their potential
residual effect while the tissue is growing. Several different antibiotics
and antibiotic combinations have been proposed. The most widely
used is triple antibiotic paste (ciprofloxacin, metronidazole, and
minocycline), which was historically introduced after trials on root
canal cultivable microflora (5052). Triple antibiotic paste has been

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found in 1 animal in situ study to disinfect 70% of root canals
compared with only 10% disinfected by 1% sodium hypochlorite
(53). However, because of the staining effect of minocycline, it was
replaced with cefaclor (54) or eliminated altogether (55). Other
strategies that prevent discoloration, such as dentin bonding (56),
may be cumbersome or ineffective. Augmentin (GlaxoSmithKline, Philadelphia, PA) was used in a recent report (57) because it has been
shown to be most effective against root canal flora (58, 59), has the
clavulanic acid that inactivates beta-lactamases that are prevalent in
endodontic infections (58), and does not discolor teeth. In the case
treated with Augmentin, despite continued normal apical maturation
of the tooth, access to the root canal to bleach the crown from discoloration because of MTA revealed no tissues in the root canal space (57).
Therefore, it is not clear to what extent repair tissue needs to be present
in the pulp space to ensure normal apical maturation in these cases.
A creamy mix of antibiotics in a powder form with water or another
sterile fluid, as is commonly advocated, results in high concentrations of
the antibiotics. These high concentrations have been recently found to
be toxic to the stem cells of the apical papilla (60). Therefore, lower
concentrations of the antibiotics need to be used, and work is currently
underway to determine the concentrations that would achieve effective
disinfection with the least toxicity to the apical regenerative tissue.
Interestingly, calcium hydroxide was not found to be toxic in the
same study (60). This medicament has been found to provide clinically
acceptable results in many case reports and case series (61, 62), and so
it provides an important alternative to be considered.

Outcomes of Alternative Therapies to Pulp

Regenerative Procedures
Endodontics has traditionally approached the problem of pulpal
necrosis by attempting to eliminate the bacteria causing the infection
and preventing recurrence by sealing the tooth against future ingress
of bacteria. Although this is highly successful in many circumstances,
teeth with immature apices present some specific challenges. Root canal
instrumentation, disinfection, and sealing are more technically difficult
to perform in such circumstances because of the tubular or reverse
tapering of the canal, together with the open apex. Furthermore,
depending on the degree of root maturation, thin root canal walls
can render the tooth weak and susceptible to fracture (21).
In the initial phases of pulp inflammation, vital pulp therapy may
be effective in maintaining the ability of the radicular pulp to continue
root formation in immature teeth (6365). However, if there is a
preoperative infection, then the current treatment options are
long-term apexification with calcium hydroxide, the MTA plug
technique, or regenerative endodontics. The long-term use of calcium
hydroxide for apexification has several disadvantages including multiple
treatment appointments, probable recontamination of the root canal
system, and weakening of root dentin, which might increase the
risk of cervical root fractures (22, 23). The long-term success of
apexification with calcium hydroxide ranged from 28%77%
depending on the degree of apical maturation in 1 classic study (21).
The MTA plug technique has been shown to be successful in terms
of healing of periapical disease (16, 17, 19, 20); however, it does not
allow for continued root development in length or width (61) and so is
not likely to strengthen the root. One large recent retrospective cohort
study examined the outcomes of MTA plug at 24 years postoperatively
(18). In this study, 252 cases were examined 110 years postoperatively; 90% of them healed, and 96% were fully functional. Using
multivariate analysis, the study showed that the presence of a
preoperative lesion and the experience of the operator were significant
predictors of outcome. Although this is an admirable outcome for this

Fouad and Verma

alternative approach to regenerative procedures, the outcome is still a

relatively small period in the lives of these children, who have a life
expectancy of 6070 years after treatment is rendered. The value of
further strengthening of the roots through regenerative procedures
remains to be determined.
The treatment approach of dental pulp regeneration has the
potential for restoring the vitality of the tooth. In immature teeth, this
can allow for continued normal physiologic development. The pulp
has the ability to promote dentin formation, which would increase
root thickness and length to prevent fracture and would potentially
also develop coronal dentin. Vital pulp has the immune response
that allows it to resist future bacterial irritation because of caries or
microleakage of restorations. Finally, a normally developed root results
in a morphology that is more appropriate for conventional endodontic
therapy if future treatment becomes necessary. Other benefits of the
pulp include the ability to repair defects because of caries and
fractures through tertiary dentin, to provide normal vascular and
sensory responses, and to promote normal function. Therefore, pulp
regeneration is an ideal treatment outcome for immature teeth
with or without preoperative infection. A retrospective review of
radiographic outcomes (61) and a recent prospective study (62)
showed that the regenerative endodontic treatment produced better
outcomes in some parameters compared with calcium hydroxide or
MTA apexification. Higher levels of evidence from randomized clinical
trials that examine multiple treatment variables are yet to be reported. As
noted previously, long postoperative periods are needed in order to
ascertain that the original goals of treatment are achieved.

Dentin Regeneration versus Repair with Other

Types of Mineralized Tissue
It is generally recognized that tissue regeneration requires an
interaction of stem cells and growth factors in a bioactive resorbable
scaffold, referred to as the tissue engineering triad (66). Current
clinical procedures appear to provide components of these elements
sufficient to produce clinically acceptable results. The published
case reports and case series have shown clinical success (4146);
however, because these are clinical case reports, it has rarely been
possible to determine the histologic nature of the regenerated tissue.
Results from in vivo animal studies using similar protocols with an
induced blood clot in the canal suggest that the regenerated tissue is not
pulp tissue but, in fact, repair tissue consisting of bone, cementum,
and inflammatory tissue (6770). There are several reports in the
literature on the histologic outcome of pulp regeneration in human
teeth (7173). In 1 of them (73), vital pulplike connective tissue
was found in the root canal of a tooth treated 14 months previously
with a regenerative endodontic procedure using platelet-rich plasma.
In another report (72), 3.5 weeks after a regenerative endodontic
procedure, more than half the canal was filled with loose connective
tissue similar to pulp tissue, and a layer of flattened odontoblasts-like
cells lined the predentin. A third report (71) found that in a molar
tooth extracted 2 years 1 month after a regenerative procedure, the
tissues formed in the canals were mineralized tissue similar to
cementoid/osteoid tissue and uninflamed fibrous connective tissue.
No pulplike tissue characterized by the presence of odontoblastlike
cells lining the mineralized tissue was observed. Finally, 1 other report
(74) revealed that a tooth treated using the currently acceptable
methods failed mechanically 26 months after treatment. Histologic
analysis revealed the presence of cementum, bone, and fibrous tissue
in the pulp space.
The clinical techniques advocated for regenerative procedures
today call for MTA to be placed in the cervical area of the tooth and
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extend into the coronal third of the root canal. This prevents mineralized tissues from forming in this area, which clearly is critical for the
mechanical strength of the tooth, and could discolor the crown (57).
Horizontal fracture in the cervical area occurred in the recent report
in which MTA was placed in this area (74). This outcome calls into
question 1 of the important goals of the regenerative procedures and
underscores the importance of undertaking randomized clinical trials
to study both healing and survival of these teeth after treatment with
alternative techniques.

The Need for an In Situ Animal Model

To predictably regenerate the dentin-pulp complex, sound tissue
engineering principles must be used. Tissue engineering models used
include the tooth slice/scaffold (75), the 6- to 7-mm long human tooth
root fragment (76), and in vivo whole pulp regeneration animal studies
(77), each of which represents a step closer to the clinical situation,
both in terms of simulating the biological response and simulating
the root canal anatomy. The findings from these studies provide proof
of concept for whole pulp regeneration, but they really do not present an
in situ model for human regenerative procedures; they would be used
on patients, particularly in cases with established infections. The reason
for this is that rodent model studies use immunodeficient animals,
which would not reject the transplanted tissues and stem cells and
are too small for in situ endodontic procedures. Larger mammals
are reasonable alternatives, but their stem cells have not been
characterized because of the absence of specific antibodies to stem cells
or dentin-specific proteins in these species. Furthermore, they may
reject xenografts of human stem cells or allografts from noninbred
members of their own species. Therefore, a good in situ model for
regenerative endodontics still needs to be identified. The ideal model
would precisely simulate the clinical situation and allow for testing of
treatment protocols and materials that could be directly translated
into a clinical trial on human patients.

The authors deny any conflicts of interest related to this study.

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JOE Volume 40, Number 4S, April 2014