Abstract

Traditional methods of treatment of immature root with necrotic pulp and apical periodontitis pose
multiple challenges. These challenges include disinfection of the root canal with standard protocols
that aggressively use endodontic files, filling the root canal with an open apex that provides no
barrier for stopping the root filling material before impinging on the periodontal tissues, and the
susceptibility of the teeth to fracture because of their thin roots. Disinfection using sodium
hypochlorite, apical barrier formation using calcium hydroxide as well as mineral trioxide
aggregate, and pulp revascularization of fractured tooth with the help of blood clot and collagen-
enhanced matrix has been discussed in detail in this article.

INTRODUCTION

Management of immature non vital teeth poses a great challenge to the clinician. Most of the
clinicians rely on traditional calcium hydroxide apexification. Calcium hydroxide induced
apexification has its own limitations such as:. [1] long term therapy for barrier formation; and,
strengthening or reinforcing of the thin fragile blunderbuss canals is not achieved. Instead, its long
term therapy has shown to make the tooth brittle due to its hygroscopic and proteolytic properties.
[2,3]
Even after apexification procedure, the clinician has to go for conventional root canal obturation
as the barrier formed is often porous and not continuous. [4,5] Mineral trioxide aggregate (MTA) has
also been used to provide an artificial barrier; however, it also has the limitations of non-
reinforcement of root canal dentin and a high cost.[6]

Recently some case reports have shown that non vital, infected immature teeth can be alternatively
treated by pulp regenerative process.[7–16] The authors have termed this regenerative process as
revascularization,[7–11] revitalization or maturogenesis.[14,15] The common aspect of all regenerative
modalities is intra canal disinfection using copious irrigation, placement of antibiotic pastes and
formation of a sterile blood clot inside the pulp cavity. The concept of pulp regeneration was first
noted in traumatic avulsed and replanted immature teeth. [16–18] Rule[19] documented root
development and apical barrier formation in cases of pulpal necrosis. The authors emphasized on
the importance of sterile blood clot and granulomatous tissue within the pulpal cavity. Various
possible explanations have been given to explain why apexogenesis/maturogenesis can occur in
these infected immature permanent teeth. These include the presence of mesenchymal stem cells
residing in the apical papilla, also known as stem cells of apical papilla (SCAP), [20] which are the
multi-potent dental pulp stem cells,[21] and resistant to necrosis/infection.[22,23] The exact etiology,
pathogenesis or histo-pathological events that occur in this regenerative process are still not known.
 Tissue engineering role in Pulp regeneration
The creation and delivery of new tissues to replace diseased, missing or traumatized pulp is referred
to as regenerative endodontics, which provides an innovative and novel range of biologically-based
treatments for endodontic disease.[1] Treatment of young permanent tooth with a necrotic root canal
[2]
system and an incompletely developed root is fraught with difficulty. Apexification enables a
calcified barrier to form at the root apex by placing a biocompatible material against the periapical
tissues via the root canal. Calcium hydroxide and mineral trioxide aggregate (MTA) have been
[3]
materials of choice for apexification procedure, but neither of them is ideal. Numerous clinical
case reports have suggested that many teeth that traditionally would receive apexification may be
[4]
treated for apexogenosis. Thus there is continuedneed to develop biologically based treatment
regimens that offer the potential for continued hard tissue formation of the young permanent tooth
with a necrotic root canal system and incompletely developed root. [2] The discovery of stem cells in
deciduous teeth elucidates the intriguing possibility of using dental pulp stem cells for tissue
engineering. [5]
It has been noted that there is a population of putative post natal stem cells in human dental
pulp.The most striking feature of Dental pulp stem cells (DPSCs) is their ability to regenerate a
dentin-pulp-like complex that is composed of mineralized matrix with tubules lined with
odontoblasts, and fibrous tissue containing blood vessels in an arrangement similar to dentin-pulp
stem complex found in normal human teeth. [6]
The post-natal pulp contains several niches of potential progenitor/stem cells, which may have
importance in mediating reparative dentine formation. Indeed, progenitor/stem cell niches are
continually being identified in all connective tissues of the body, where they play a fundamental
role in wound repair processes. This subset of undifferentiated cells can represent as little as 1% of
the total cell population. However, they produce multiple highly differentiated progeny in response
[7]
to specific extracellular signals. Central to the niche is the 'true' adult or 'mother' stem cell which
displays an infrequent, yet almost unlimited self-renewal. At mitosis, these cells give rise to a
renewed mother stem cell and a daughter transit amplifying progenitor cell. These daughter
progenitor cells possess a more limited capacity for self-renewal, but are highly proliferative. They
also appear to control multi-potentiality, and are capable of following along several cell lineages to
ultimately produce terminally differentiated cells such as osteoblasts, odontoblasts, and, adipocytes,
chondrocytes, and neural cells. [7]
  Stem Cells

A stem cell is defined as a cell that can continuously produce unaltered daughters and, furthermore,
has the ability to generate cells with different and more restricted properties. Stem cells can divide
either symmetrically (allowing the increase of stem cell number) or asymmetrically. Asymmetric
divisions keep the number of stem cells unaltered and are responsible for the generation of cells
[8]
with different properties. These cells can either multiply progenitors or transit amplifying cells.
Types of stem cells
[9]
The stem cells were first suggested by Danchakoff and by Sabin and Maximow.

Stem cells can be classified according to:

 Potential for differentiation into totipotent, pluripotent, multipotent, and unipotent cells
 The tissue of origin for embryonic /adult stem cells
 Their capacity for tissue re-population in vivo in short, medium or long time regeneration.
Different types of stem cells are described in [Figure 1] and [Figure 2].

Types of stem cells

Types of dental pulp stem cells

Dental pulp stem cells
Dental pulp entrapped within 'sealed niches' of the pulp chamber, is an extremely rich site for stem
cell collection. These stem cells are called DPSCs when found in adults and stem cells from human
exfoliateddeciduous (SHEDs). [5],[10]
SHED
The use of SHED for tissue engineering might be more advantageous that that of stem cells from
adult human teeth; they were reported to have a higher proliferation rate than stem cells from
permanent teeth. [5] They are ideally suited for young patients at the mixed dentition stage who have
[11]
suffered pulp necrosis in immature permanent teeth as a consequence of trauma. The main task
of these cells seems to be formation of mineralized tissue which can used to enhance orofacial bone
regeneration. [12]
Stem cells from apical papilla
[13]
SCAP were recently discovered by Sonoyama. SCAP exhibits a higher proliferative rate and
[9]
appears more effective than PDLSC for tooth formation. Compared with DPSC, SCAP have a
great numbers of STRO-1 positive cells, faster proliferation, a greater number of population
doublings and increased capacity for in vivo dentine regeneration. [14]
Periodontal ligament stem cells
[15]
Presences of multipotent post natal stem cells in human PDL were first described by Seo. They
can be isolated from Cryo-preserved periodontal ligaments while maintaining their stem cell
characteristics, including the expression of MSC surface cell markers, single-colony-strain
generation, multipotential differentiation and cementum/periodontal-ligament-like tissue
regeneration, thus providing a ready source of MSCs. [16]
Apexification and new trends of regenerative endodontics
Apexification is a procedure to promote the formation of an apical barrier to close the open apex of
an immature tooth with a nonvital pulp such that the filling material can be contained within the
root canal space. [17] Maturogenesis is a more appropriate term than Apexification, because not only
[18]
the apex but the entire root is allowed to mature as in non-traumatized tooth. The use of term
"Revascularization" was adopted by Iwayato describe the clinical healing of periapical abscesses
and continued root formation in immature teeth with nonvital pulps. [19]
The clinical decision whether to perform apexogenesis or apexification for immature teeth appears
to be clear cut with teeth deemed to have vital pulp tissue are subjected to apexogenesis and teeth
[20]
deemed to have non vital pulp tissue receives Apexification. Calcium hydroxide and mineral
trioxide aggregate (MTA) have been materials of choice for Apexification procedures, but neither
materials is ideal. A number of shortcomings can be summarized as for Ca (OH) 2 apexification:
1. Long time span for entire treatment;
2. Multiple visits with heavy demands on patient and carers and inevitable clinical costs;
3. Increased risk of tooth fracture using Ca(OH) 2 as a long term root canal dressing. [21]
However MTA has provided more favorable results and improved patient compliance. Pulpal tissue
preservation, uniform calcified bridge formation, rate of bridge formation and providing excellent
[22]
seal make MTA a new choice for Pulpal maturogenesis. From a physiological point of view
calcified material benefits vs. regeneration in Apexification is ongoing point of debate as calcific
[23]
metamorphosis is a degenerative disease. The advantages of pulp revascularization lie in the
possibility of further root development and reinforcement of dentinal walls by deposition of hard
tissue, thus strengthening the root against fracture. [24]
Two types of pulp regeneration can be considered based on the clinical situations:
1. Partial pulp regeneration
2. De novo synthesis of pulp. [20]
Ostbypostulated the tissue reorganization in the canal space filled with blood clot. It was observed
that tissue formed in the canal was not pulp but granulation or fibrous tissue and in some cases the
ingrowths of Cementum and bone growth.It has been experimentally shown that apical part of pulp
may remain vital and, after reimplantation, may proliferate coronally, replacing the necrotized
[25]
portion of pulp. Successful regeneration depends on race between the new tissue and bacteria
populating the pulp space is strengthened by the fact that the incidence of revascularization is
enhanced if the apex shows radiographic opening of more than 1.1 mm and the tooth is replanted
within 45 min (thus increasing chances of revascularization by 18%). [26]
Pulp tissue engineering and shift of treatment protocol
Before isolation of DPSCs, pulp regeneration was tested using Modern tissue engineering concepts
by growing pulp cells onto synthetic polymer scaffolds of polyglycolic acid (PGA) and in vitro and
[27]
in vivo analysis performed. These approaches are basically a proof of principle to test whether
cultured pulp cells can grow well and produce matrix on PGA, and whether the engineered pulp can
be vascularized. [28]
While vascularization is a universal issue for an engineered tissue, it is of special concern for pulp
tissue engineering b'coz of the lack of a collateral source of blood supply. Use of angiogenic factors
inducing factors such as vascular endothelial growth factors (VEGF) could enhance and accelerate
[28]
pulp angiogenesis. Regeneration of tissue into the apex of an immature permanent tooth may
come from stem cells already residing in vital pulp tissue, the apical papilla, PDL or alveolar bone.
[29]
Stem cells have been identified in greater number within the PDL of diseased teeth where the
inflammatory process actively recruited immature cells. [30]
Mooney first described technique to engineer new pulp-like tissue from cultured human pulpal
fibroblast. Regeneration of pulp or periodontal tissues relies on the provision of appropriate
biodegradable scaffolds which are capable of containing or being seeded with growth factors and
[31]
bioactive signaling molecules, supporting cell organization and growth of vascular supply. Yet
no matrix has been proved ideal; collagen and polymer scaffolds are able to support in vitro survival
of DPSC and PDLSC. [32] Cordeiro seeded SHED and endothelial cells onto biodegradable scaffolds
within human tooth slices then implanted them into immunocompromised mice. It was observed
that cells differentiated into odontoblast like and endothelial like m in vivo resulting in tissue
closely resembling dental pulp with a viable blood supply. [33]
Management of immature teeth: A new perspective
Clinical reports show that after conservative treatment severely infected immature teeth with
periradicular periodontitis and abscess can undergo healing and apexogenesis and maturogenosis.
Regenerative endodontics promotes a shift in protocol for treating endodontically involved
immature permanent teeth by conserving any dental stem cells that might remain in the disinfected
viable tissues to allow tissue regeneration and repair to achieve apexogenesis/maturogenisis.
Even though there exists no evidence based guidelines regarding cases that can be treated with
conservative approach recent case reports have shown that the traditional approach of apexification
may compromise certain cases that have the potential to undergo apexogenisis. Several reports have
documented this observation. These case reports have generated interest in understanding the
observation that important type of cells and tissues must have survived after disinfection thereby
allowing the root to undergo maturation. Example of HERS and SCAP in the apical papilla. SCAP
have been proposed to the cell source of root odontoblasts. [13]
 Dental pulp regeneration
Dental pulp is the innermost and the softest tissue of the teeth. It has various functions namely
nutritive, formative, protective and reparative. Pulp is the only vascularized tissue covered in highly
mineralized structures like dentin, enamel and cementum. It also maintains the homoestatis of the
tooth. [1] Endodontic treatment or root canal therapy for irreversible pulpitis is pulpectomy,
involving pulp extirpation followed by root canal enlargement and obturation with gutta percha, a
bioinert thermoplastic material. [2] A substantial amount of tooth structures, including enamel and
dentin, is removed during endodontic treatment, potentially leading to posttreatment tooth fracture
and trauma[2,4]. Endodontically treated teeth have lost pulpal sensation, and are deprived of the
ability to detect secondary infections. [2,3,5] The complications of current endodontic treatment are
inevitable because of pulp devitalization or the loss of the tooth’s innate homeostasis and defense
mechanisms. Pulpectomy, the most common endodontic treatment, involves extirpation of dental
pulp, and therefore leaves no dental pulp stem cells in the same tooth for pulp regeneration. For a
patient who requires
endodontic treatment in a given tooth but has intact dentition otherwise, no healthy tooth is to be
sacrificed for isolation of dental pulp stem cells. Even in patients whose autologous dental pulp
stem cells can be harvested, for example, from extracted wisdom teeth, clinical therapy of dental
pulp regeneration is difficult to develop due to excessive costs, including cell isolation, handling,
storage, and shipping, ex vivo manipulation, immune rejection (for allogeneic cells), not to mention
liabilities of potential contamination, pathogen transmission, and tumorigenesis that may be
associated with cell transplantation . [14,15]
Cell homing has been regarded as a process of exit of hematopoietic stem cells from blood vessels
by transendothelialization and subsequent migration. [16]. In
tissue regeneration, cell homing is dubbed as active recruitment of endogenous cells, including
stem/progenitor cells, into an anatomic compartment . [17,18] Particular discussion on this topic has
been made to determine whether dental-pulp-like tissue can be regenerated in endodontically treated
root canals of real-size, native human teeth by chemo taxis-induced cell homing, rather than cell
transplantation. Our motivation for the study is to explore whether chemo taxis-induced cell homing
is sufficient for the regeneration of dental-pulp-like tissue in endodontically treated root canals of
real-size, native human teeth.
USAGE OF VITAL PULP
It is also essential to know the value of vital pulp in a fully formed tooth. Endodontic disease is
apical periodontitis, and as such, the biologic rationale for endodontics is the prevention or
treatment of apical periodontitis. For apical
periodontitis to be present, the root canal must contain a necrotic infected pulp .[19] Therefore, the
noninfected pulp ensures no apical periodontitis. Thus, maintaining the vital pulp prevents apical
periodontitis, and the potential to
regenerate an injured or necrotic pulp would be the best root filling possible.
REGENERATIVE POTENTIAL OF DENTAL PULP
Exposed Pulp
Treatment of the exposed pulp remains quite controversial, with different approaches endorsed by
different dental specialties. Vital therapy on traumatically exposed pulps is very successful [21],
whereas vital pulp therapy on the cariously exposed tooth is not nearly as successful . [22] The
difference in success rates is explained by the status of the pulp at the time of the procedure .
Capping the healthy pulp gives very high success rates, whereas capping the inflamed pulp results
in lower and less predictable success. [23,24,25] On the other hand, with a carious exposure the area
and depth of inflammation are very unpredictable, and pulp capping at the superficial exposure site
is popular. Thus, it is very likely that we would be capping an inflamed pulp, and more failures
(necrotic pulps) would result.
Another extremely important factor in the success of treating a vital exposure is the coronal seal
after the pulp capping/pulpotomy. [26] Cox et al. showed that the pulp
can withstand the toxicity of most dental materials, and that what was previously interpreted as
toxicity was, in fact, due to the material not sealing adequately. Therefore, it is considered essential
that a well-sealed coronal seal be placed over the vital pulp therapy. This is considered much more
important than the material used on the vital pulp.
Unexposed Pulp
The inflamed pulp which is unexposed by caries or trauma has the ability to be repaired. Although
our diagnostic ability to differentiate a vital from a necrotic pulp is good, differentiating between
reversibly and irreversibly inflamed pulp remains an educated guess at best. [20] However, the
younger the pulp is, the better will be its repair potential.
REGENERATION OF ROOT CANAL CONNECTIVE TISSUE
The connective tissue remaining in the root canal initiates various defense responses because of the
presence of bacteria. The endodontic community has mainly focused its research on root canal
disinfection and subsequent filling
with an inert material. There is a large percentage of failures associated with periapical disease on
badly treated teeth. Since root canal treatments are technically difficult to complete, especially by
non-specialist clinicians, endodontics would benefit from alternative approaches [27].Various
research have been done to regenerate a vascularized tissue in an empty canal; however, the absence
of infection in these research has limited their relevance to the clinical situation [28]. Subsequently,
the endodontic community has largely focused on more mechanical aspects of root fillings,
including the use of disinfection
methods, files and instrumentation, and alternative filling materials and techniques. For more than
30 years, little progress has been made on the design of new approaches in endodontics, other than
shaping, disinfecting, and novel filling methods. Over the past decade, revascularization of the root
canal has been re-proposed with a two-visit therapeutic approach [29]. In this procedure, the root
canal system is disinfected with a mix of antibiotics, and a blood clot is subsequently induced in the
canal itself by irritation of the periapical apex area with an endodontic file. This clot is then
protected by a mineral trioxide aggregate plug, and the coronal cavity is sealed and restored by
conventional treatment. Although case reports have been published based around this approach few
have attempted to describe the regenerative processes taking place[30].
Although this was initially presented as a regenerative technique, indicating that the regenerated
connective tissue was a dental-pulp-like tissue, many authors have described it as a
revascularization approach rather than a regenerative one, meaning that there is no histological
proof that the new tissue forming in the root canal is comparable with the pulp. Recently, Wang et
al. described a combination of dentin, cementum, and pulp regeneration in a dog tooth following the
use of a revascularization approach [31]. So far, our limited knowledge regarding the healing
process has limited the development of new techniques. The hypothetical involvement of SCAP
cells remaining viable even in very aggressive infection conditions implies a limitation to this
therapy in immature necrotic teeth [32].
However, further work on SCAP cell use in this area is required. Many other scientific/clinical
questions still remain in this area. While regeneration of the whole dental pulp would be ideal
clinically, regenerating a connective tissue, mineralized or not, might provide an acceptable
compromise. Indeed, the aim of our current research is to prevent any further infection of the tooth
and protect the
periapical tissues. The inert materials used in endodontics have limited sealing ability and,
ultimately, a limited clinical longevity.
CLINICAL ADVANTAGE OF PULP REGENERATION
Although the success rate of endodontic treatment is relatively high (78–98%) [33-36] ,there are
many problems associated with this aggressive treatment, which includes the following:
 Mostly Endodontic procedures are technically sensitive; mishaps occur such as blockage of the
rootcanal space, and breakage of instruments in the canals and create perforations [37 -42] which
will leave infected tissue behind or even cause loss of the tooth;
 If the teeth is immature that has little dentin structure after loss of pulp tissue, endodontic
treatment cannot prevent its susceptibility to fracture from traumatic
injuries [43-47].
 Teeth after undergoing endodontic treatment lose a significant amount of tooth structure. Post-
space plus crown preparation sacrifices more tooth structure,
which weakens the tooth [48-50].
 Pulpless teeth have no sensation to irritations, rendering caries progression unnoticed by
patients.
Teeth loss is higher for endodontically treated teeth than nontreated teeth owing to secondary caries
and complex restoration-associated problems [51-54].
Surgical procedure

A sequence of illustrations describing step-by-step the sequences of the clinical study, including
caries treatment with composite resin wall restoration followed by pulpectomy, cell processing, and
cell transplantation, followed by final restoration. CBCT cone beam computed tomography, CPC
Cell Processing Center, GMP good manufacturing practice, MDPSC mobilized dental pulp stem
cell, MRI magnetic resonance imaging
 NANO-TECHNOLOGY AND PULP REGINRTATION

Different applications of Nanotechnology in pulp regeneration and how they act

as assisting factors in the success of pulp regeneration and revascularization
Nano-scaffold
Construction of Nanofibrous and microporous scaffolds are very promising to promote dental pulp
regeneration as a mimetic extracellular matrix. By electrospinning, matrices of different synthetic
and natural polymers are built, with nanofibers of diameters closest to the size of collagen
nanofibers (50 to 500 nm). The electro spun randomized nanofiber network and the created
micropores (diameter inferior to 100 µm) mimic the pattern of the connective tissue matrix allowing
tissue regeneration to occur successfully. Capacity of these matrices to be functionalized may also
allow success in the different steps of the complex endodontic regeneration.
Examples for Nano-scaffold, the use of poly (L-lactic acid) (PLLA) Nano-fibrous microspheres
(NF-MS), Poly (lactide-coglycolide)-polyethylene glycol (PLGA-PEG) Nano-particles, the
synthetic polysaccharide hydrogel, VitroGel 3D,these types have the ability to induce the bone
formation and differentiation of odontoblasts and differentiation of SCAP, also help in rapid
periapical healing observed with the follow up 18-24 months .
Nanotechnology for the design of artificial stem cell niches
Nanotechnology can be used to make an artificial microenvironment that will direct stem cells or
progenitor cells towards a precise fate and function. Extracellular matrix (ECM) molecules
represent the noncellular components of steam cells niches and are important for the creation of a
particular microenvironment. Providing ECM in nanoscales allow cell adhesion and immobilization
of signals molecules affecting the fate and behavior or steam cells .
Nanomaterial for pulp medication
To build biomaterials at the nanoscale level is very crucial for dental pulp regeneration as It allows
the concentration of many different functions in a small volume and presents the advantage of
increasing the quality of targeting while controlling the cost and delivery of the active molecules.
These endodontic also due to their size, have the ability of rapid dispersion into the hard-to-reach
spaces in the complex root canal system which leads to a better antimicrobial effect .
Nano-irrigantes
Nanotechnology enable us to get irrigants with advanced and improved properties .
 Silver nanoparticles have favorable ability in removing the smear layer and it's the material of
choice for the removing e-faecalis bacteria from root canal. the cytotoxic effect of silver is
disappeared when used as nanoparticles. In Comparison with NaOCl observer that the novel
 irrigant, which was based on silver Nano particles, was as effective as NaOCl 5.25% in
elimination of both E. Faecalis and S.Aureus.
 Nano MgO irrigant with 5 mg/lit concentration showed notably long-term efficiency with
regard to removal of E. Faecalis in comparison with NaOCl.
 New Nano chitosan irrigant showed superior smear layer removal and penetration. High
permeability and immediate spread over dentin layer.
Nano instrument
Nanomaterials, with a smaller size, are being suggested for surface modification and reduction the
incidence of failure in the rotary nickel-titanium files .
Nanotechnology in endodontic sealers
Nano-endodontic sealers having the ability to seal the obturating material with less space inhibiting
the bacterial biofilm on the dentinal tubules .
Nano-obturating materials
The incorporation of nanoparticles in the obturating materials may enhance the sealing properties
and antimicrobial efficacy .
Nanorobots and Nano-terminators are also new technologies for local anesthesia with fewer side
effects and complications .
Nano microscopes:
As pulp regeneration is a very sensitive technique, so improving the vision affect greatly the
success of technique .
Nano radiography:
Digital radiographic imaging aiming to reduce the radiation dose and improve the quality of the
images
 Clinical procedure for pulp-dentin regeneration

The approach of regenerating dental pulp tissue by inducing blood into the root canal was first
proposed by Ostby in the 1960s14 and then abandoned for over 20 years with no obvious outcome.
In the 1970s, the understanding that revascularization, or reestablishment of a vascular network
within the root canal, is essential for the completion of root development, came to the fore, having
originated from traumatology.15,16 The term, revascularization, was then used in initial case reports
on regenerative endodontic therapies.3,17 During the period of more than a decade that followed the
first case report, various protocols for pulp-dentin regeneration were introduced. This new treatment
modality was adopted by the American Dental Association in 2011. However, evidence-based
guidelines that provide the most favorable results have not yet been fully established, but are being
gradually developed, based on many clinical and basic research studies.

Differences from conventional endodontic treatment

Similar to conventional root canal therapy, the primary goal of REPs as an endodontic treatment is
the resolution of apical periodontitis. However, there are certain differences in the basic concept and
related procedures. First, REPs are originally applied to immature permanent teeth, with thin walls
and wide-opened apices. Aggressive filing is performed for infection control in endodontic
treatment. However, in REPs, mechanical debridement using endodontic files is contraindicated to
avoid further weakening of the thin root canal wall and to protect the vitality of apical tissue stem
cells.17,18 Instead, sufficient chemical disinfection using an irrigant and intracanal medicaments is
proposed.
Second, disinfection in REPs should be performed with thorough consideration to cell cytotoxicity.
Even though sterile environment may be achieved by sufficient disinfection, pulp tissue
regeneration requires a balance between disinfection and the microenvironment necessary for cell
viability, in order to induce stem cell survival and differentiation. Various concentrations of sodium
hypochlorite (NaOCl), ranging from 0.5% to 6 %, have been used for disinfection. 19 Recently,
several studies have reported that concentrations of NaOCl higher than 3% may exhibit cytotoxicity
toward stem cells of apical papilla (SCAP) and interfere with cell adhesion on the dentin surface. 20–
22
For these reasons, recent studies including clinical considerations of the American Association of
Endodontists (AAE) recommend the use of lower concentration of NaOCl in REPs. In the same
vein, the use of lower concentrations of triple antibiotic paste (TAP) or calcium hydroxide were
recommended as intracanal medicaments.23
Finally, REPs utilize tissue engineering to form a pulp-dentin structure in the canal. Intracanal
bleeding has a positive impact on the three requirements for tissue engineering: stem/progenitor
cells, scaffolds, and growth factors.10,24,25 It is proposed that inducing bleeding into the canal results
in the delivery of mesenchymal stem cells (MSCs) to the site. 25 The blood clot that forms acts as the
scaffold, as well as a rich source of growth factors that may play an important role in the
regeneration process.10,26 Since a recent study reported that ethylenediaminetetraacetic acid (EDTA)
solution may release various growth factors entrapped in dentin, thereby promoting differentiation
of dental pulp stem cells (DPSCs) into odontoblast-like cells, EDTA has been recommended as the
final irrigation.27

Clinical protocol: revascularization

REP is performed within the principles of conventional endodontic treatments, but with some major
changes, such as a disinfection process which totally relies on chemical irrigation (while taking into
consideration cytotoxicity toward cells to be recruited for the canal), as well as the stimulation of
pulp-dentin regeneration via bleeding induction. The currently recommended procedures are as
follows (Figure 1).24,28

Figure 1. Schematic illustration of revascularization procedure. Revascularization is considered for

immature teeth with open apices, pulp necrosis, and apical periodontitis (a). After accessing the
opening (b), gentle irrigation limited to coronal part of the chamber is performed. A radiograph with
K-file insertion (c) provides the approximate tooth length, which helps to determine a working
length. Low concentration of NaOCl (1.5 or less than 3%, 20 mL/canal, 5 min) is used for
disinfection (d), following which saline or 17% EDTA is used. After copious irrigation and canal
drying with paper point (e), intracanal medicaments, such as Ca(OH) 2 or TAP were placed, and
covered with temporary filling material (f). After confirming the absence of any signs of infection,
the final step is initiated. Final irrigation is performed with sterile saline and 17% EDTA (g). After
the canal has dried (h), a pre-curved K-file is introduced 2 mm past the apical foramen and rotated
to induce bleeding (i). Blood fills the canal from the bottom and the blood clot can be identified
after 15 min (j). After the blood clot is confirmed, capping materials such as MTA are placed over
the blood clot (k). Regeneration of pulp-dentin leads to root development with thickening,
lengthening, and apical closure, as well as maintenance of tooth vitality (l).

First visit

All visits, except for the final visit, are designed with a focus on disinfection of the root canal. After
local anesthesia, rubber dam isolation, and access, gentle irrigation which is limited to the coronal
part of the pulp chamber is performed. It is recommended that the canal be inspected using a dental
microscope to confirm the presence of residual vital tissue and the level to which it is present. 24 A
K-file, or alternatively a gutta-percha cone, should be introduced into the canal to establish a
working length.11,29 In case when inserting a file into the canal, a little resistance caused by viable
tissue or pain sensation is reported, a file should not progress deeper. A file could be fixed with wax
or cotton pellet during radiographs taken (Figure 1(c)).
Removal of necrotic tissue and the disinfection of the canal were accomplished by gently irrigating
the canal with a minimum 20 mL NaOCl (Figure 1(d)). Lower concentrations of NaOCl are
recommended (1.5 or under 3%, 20 mL/canal, 5 min). 20,30 The canal is then irrigated with saline or
17% EDTA (20 mL/canal, 5 min). The needle should be positioned at a point 2 mm short of the
apical foramen in order to minimize cytotoxicity to apical tissues. Negative pressure irrigation
procedures such as EndoVac (Discus Dental, Culver city, CA) may be considered. 31 Mechanical
debridement is contraindicated so as not to weaken the root wall.
After the canal is disinfected with copious irrigation and dried with paper points (Figure 1(e)), it is
recommended to place intracanal medicaments, either calcium hydroxide (Ca(OH) 2) or TAP using a
lentulo spiral or syringe. Treatment with TAP (a 1:1:1 volume combination of ciprofloxacin,
metronidazole, and minocycline) at a low concentration (0.1–1.0 mg/mL) is recommended to lower
cytotoxicity toward stem/progenitor cells. Double antibiotic paste (DAP) without minocycline may
be considered if avoidance of tooth discoloration is desired. In order to prevent coronal leakage of
bacteria, a sterile cotton pellet may be placed over the medicaments and the pellet covered with 3–4 
mm of temporary filling material, such as Cavit (3M ESPE, St Paul, MN), IRM, and glass ionomer.
Interim visit

The patients could be recalled within a time interval of 1–4 weeks. If clinical signs and symptoms
persist, the disinfection procedures implemented during the first visit should be repeated.

Final visit

After confirming that signs of persistent infection are absent, the tooth is anesthetized with 3%
mepivacaine without vasoconstrictor, and the temporary restoration removed following rubber dam
isolation. Copious and gentle irrigation with sterile saline or 17% EDTA should be repeated until no
medicament is evident in the canal (Figure 1(g)). After the canal is dried with paper points, bleeding
is induced by over-instrumenting with K-file. A pre-curved K-file is introduced 2 mm past the
apical foramen and rotated to induce bleeding below a point approximately 3 mm apical to the
cemento-enamel junction (CEJ; Figure 1(i) and (j)). The time estimated for blood clot formation is
15 min.3,29,32 The stability of the blood clot could be confirmed using the reverse side of a paper
point. An alternative method of inducing a blood clot is the use of platelet-rich plasma (PRP) or
platelet-rich fibrin (PRF). After stability of the blood clot is confirmed, mineral trioxide aggregate
(MTA) cement is placed over the clot as capping material. A 3–4 mm layer of MTA is
recommended. In order to minimize apical displacement of MTA, resorbable matrix, such as
collagen matrix, may be placed over the blood clot. In case MTA with a long setting time is used,
final restoration is performed during the next visit.
METHODS OF ISOLATION DURING ENDODONTIC TREATMENT

Despite the many advantages of dental dams, they are often underutilized in endodontic
and restorative procedures. only 44% of respondents reported using dental dams all of the time
during root canal treatment.1 Another article cited only 60% usage among general practitioners
surveyed.2 These numbers may not surprise endodontists or educators who have been treating
patients and teaching endodontics for years. In fact, a 1967 publication indicated that merely 7% of
respondents used dental dams for endodontic treatment.3

FIGURE 1. This endodontic file is lodged in the oesophagogastric junction; a dental dam was not
used during endodontic treatment.5

While there are any number of rationalizations for not using a dental dam for patient care, much less
endodontic treatment,4 what some clinicians fail to consider are the innumerable benefits — and not
just for endodontic therapy, but also for restorative treatment. These include protection from
aspiration and swallowing of instruments 5 (Figure 1), burs, solutions, restorative materials, debris
and particulates. Besides providing isolation and moisture control (thus reducing airborne
pathogens), dams improve access to and visualization of the treatment site. They also aid tongue
and cheek management while improving patient comfort and enhancing clinical efficiency.
Initially made available to dentistry, royalty free, by Sanford C. Barnum, DDS, in 1862, today’s
dental dams are listed by the U.S. Food and Drug Administration as Class 1 medical devices. 6 The
use of dams for endodontic treatment is defined as the standard of care by the American Association
of Endodontists.7 Even so, marginal use of dental dams remains a concern in dental practice — even
though every dental school in the United States teaches use of the dental dam.
DENTAL DAMS IN PERSPECTIVE

Too often in the teaching clinic, dental dam placement seems to take on the burden of a
comprehensive treatment plan, and unnecessary time is spent to achieve a perfection that is not
needed. Voltaire popularized the aphorism “Perfect is the enemy of good” in 1770. This was hardly
unique, as the concept was also espoused by others, including Aristotle and Confucius.8

My approach to using dental dams came as a general practitioner as I was developing an interest in
endodontics. Having spent a day in the office of an endodontist who would later become my
graduate mentor, I saw that, contrary to most textbook recommendations, he would routinely isolate
more than one tooth (Figures 2A through 2D). I emulated his technique and was soon routinely
applying dams for restorative procedures, including crown and abutment preps — and, of course,
endodontic therapy. With increased frequency came increased proficiency and efficiency.

FI
GURES 2A through 2D. Field isolation with (A) and without (B) a dental dam in the embrasures;
(C) isolation of a bridge using two clams and putty; (D) high-end isolation with putty sealer.
FIGURE 2C COURTESY IVAN NEDELTCHEV, DDS; FIGURE 2D COURTESY DALAL AL
AMIR, BDS

In placing a dam for a simple restoration, a fast-acting anesthetic would be used, followed by
isolation, placement of the saliva ejector, and, almost immediately, initiation of the preparation.
With the proper use of a dam, vision of the operating field, tongue blockage and cheek retraction
are no longer issues.
It is common for dental schools to teach a high degree of precision when it comes to placing dental
dams. Stamps are often used to guide hole placement (a technique in which small holes are punched
to optimize the creation of a seal around teeth) and the dam may be inverted around the teeth with
specialized instruments to facilitate the seal. Commonly, the dam clamp is placed on the tooth first
and the rubber dam stretched over the bow of the clamp and adapted to the teeth. Technique-wise,
this can prove challenging.
CLINICAL APPROACH TO PLACEMENT

Dental dams are available in various thicknesses and colors (and, of course, in rubber and non-latex
materials). Generally, heavy or extra heavy material is used in schools, and some practitioners
choose heavy materials to help retract soft tissues. Others prefer thin and light-colored (or white)
dams as being easiest to work with. Fresh rubber dam material does not tear, and limited quantities
should be ordered and stored in a refrigerator. It is worth the extra cost to avoid chairside
aggravation. Care needs to be taken regarding latex allergies, so non-latex dams should be
available; these can be made from polyethylene, polyvinylchloride or nitrile.

FIGURE 3. Only a few dental dam clamp styles are needed for most applications.

FIGURE 4. During emergency treatment, time and options are sometimes limited. As shown here,
the gingivae and alveolus are clamped through the dental dam; this was followed by cleaning,
shaping and drainage.

When placing a dental dam, clamps are retained by engaging the tooth below the height of contour.
Clamps should be selected that have wings and most closely approximate the size of the tooth
(Figure 3). Although clamp designations vary among manufacturers, the basic armamentarium
should consist of winged clamps, butterfly-type clamps for anterior teeth, universal premolar
clamps and molar clamps. As expertise develops, the practitioner may accumulate a broader
inventory of clamp styles. Some situations require ingenuity. During emergency visits, for example,
where crown lengthening is not possible, the alveolus is sometimes clamped (Figure 4); other times,
slots are used for placement (Figures 5A and 5B).

FIGURES 5A and 5B. A slot is used to help with isolation for removal of the
crown on the canine and eventual treatment.

Using a dam punch and the largest sized hole makes placing the holes over teeth easier and helps
minimize tearing, especially if using fresh dam material (Figure 6). Punch the hole(s) in the middle
of the dam. If using field isolation, punch multiple holes approximately 5 mm apart and in an arc (if
appropriate). Placing the bow of the clamp through the hole and engaging the clamp wings
eliminates the need for floss on the clamp because the clamp is always outside the dam. When
clamps fail, the bow usually breaks; therefore, unless both sides of the clamp have floss, this
practice can be considered unwarranted.

FIGURE 6. Using a dental dam punch and the largest appropriately sized hole makes placing the
dam easier and helps prevent tearing.

Use a plastic frame if performing endodontic therapy (or stainless steel frame for restorative
treatment), and (1) place the clamp and dam, isolate the tooth (or teeth), and attach the frame; or (2)
place the clamp/dam/frame, isolate the teeth and adjust the frame (Figure 7). If a tooth is tight, place
one hole over two teeth, or simply cut the strip between two holes and place over two teeth. A
single hole can be stretched over two teeth, as well. Manufacturers also offer alternative
combinations of dam frames, shapes, materials and formulations that may suit an operator’s
preference (Figure 8).

FIGURE 7. The rubber dam, clamp and frame can be carried to the mouth as one
unit, and adjusted. The use of floss is optional.

FIGURE 8. This patient reports anxiety with masks or anything covering his nose. The use of an
alternative dam kept the appliance below his nose and eyesight. With periodic breaks, isolation was
maintained and treatment appropriately rendered.

FIGURE 9. Field isolation and a thin, light dental dam allow easy placement of working films.
Note how the dam is folded under and away from the patient’s nose.

Rearrange the dam on the frame so it is comfortable for the patient and provides the access needed
for the procedure. Folding the top of the dam and keeping it below the patient’s nose is preferable to
cutting the material around the nose (Figure 9). Place the saliva ejector and/or give the patient
control over its placement and use; with a properly placed dam, the saliva ejector will not get in the
way. If the dam interferes when managing gingival margins, cut it (as the dam is there to help and
not hinder).

The key point is that isolation with a dam is significantly better than if not using one. In addition, if
leakage is detected, putties and caulks are available to quickly improve the seal.
Conclusion

Stem/progenitor cell-based tissue engineering and bioprinting are promising approaches to protect
the vitality and restore the integrity of dental tissues. Many attempts proved to be very promising, as
reported in various in vitro studies, animal studies, and very few human trials. Despite the fact that
the proposed biomaterials and techniques could be promising for future dental tissues’ regeneration,
still the complexity and the multicellular interactions naturally existing in dental structures represent
great currently unsolved challenges. A clear set of universally accepted markers for the isolation
and characterization of stem/progenitor cells and the development of serum and animal product-free
culturing media for cell expansion are further major hurdles prior to considering stem/progenitor
cell-based transplantation therapies for routine clinical application. Finally, the side effects of
stem/progenitor transplantation should be clearly investigated, prior to becoming a clinical
therapeutic reality in restorative dentistry.