Received : 01‑09‑15

Review completed : 18‑11‑15

Review Article Accepted : 29‑11‑15

REGENERATIVE ENDODONTICS -TREATMENT OPTIONS AND

CHALLENGES TO SUCCESS

Vivek Chand CU, * Sam Joseph VG, ** Jinu George, *** Mini K John, † Anand S, ††
Mali G Nair †††

* Post Graduate Student, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Karnataka,
Kerala, India
** Professor & Head, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Karnataka,
Kerala, India
*** Senior Resident, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Karnataka,
Kerala, India
† Assistant Professor, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Karnataka,
Kerala, India
†† Post Graduate Student, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum,
Karnataka, Kerala, India
††† Professor, Department of Conservative Dentistry and Endodontics, Government Dental College, Trivandrum, Karnataka, Kerala,
India
_________________________________________________________________________
ABSTRACT The concept of regaining the vitality of dental
When maintenance of tooth in a vital condition pulp as a potential treatment option has astounded
is no longer possible, the traditional treatment the endodontic community.[2] Since then, for teeth
philosophy advocates endodontic treatment to identified to have a favorable response to the new
retain it in a functional state. A paradigm shift treatment, there has been a paradigm shift from
of this thinking occurred with routine root canal therapy to vital pulp therapy,
revascularization/regenerative procedures. initiating an era of pulp
Dental pulp is a specialized tissue organized in revascularization/regeneration. The tissues of
an order spatial arrangement and regenerating interest in regenerative endodontics include
it is somewhat exigent. With advancements in dentin, pulp, cementum and periodontal tissues.[3]
molecular science and tissue engineering, the The new tissue formed has been described
approach is becoming being refined with variably as pulp-like, dentine-like or cementum-
higher success rates. This review article will like and the literature is replete with various
detail some of the methods of regeneration methodologies of achieving this outcome. The
and the challenges to achieve success in these message from these sources often is ambiguous -
procedures. does regeneration or revascularization of the pulp
KEYWORDS: Pulp regeneration; actually takes place? This article reviews the
revascularization; pulp stem cells; tissue literature on the various approaches to achieve
pulpal regeneration and the challenges to achieve
engineering; scaffolds; pulp vitality
success in the treatment.
PHILOSOPHY OF REGENERATION
INTRODUCTION
Dental pulp is a unique and self repairing tissue
Advances in molecular biology and stem cell
composed of fibroblasts, progenitor cells,
technology have resulted in the emergence of
vascular cells, nerve cells and immune cells. Like
regeneration as a new treatment modality in
any other injured tissue in the body, pulp also
medical science. Regenerative medicine is
mounts an initial defense response in an attempt
described as a process of replacing, engineering
to remove the infection and favor wound
or regenerating human cells, tissues or organs to
healing.[4] A layer of odontoblast cells line the
restore or establish normal function.[1] A quarter
pulp chamber in a spatial order which maintains
century after its inception, several treatment
the dentine formation equilibrium as long as pulp
procedures which earlier focused on replacement
is vital. Any assault to the pulp causes disruption
with artificial structures have been substituted
of this balance. In revascularization procedure, a
with regenerative procedures.
blood clot is intentionally created to form a

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scaffold on which regeneration can occur, to a with the ability to alter the physical and chemical
great extent in the identical manner as any natural properties. They are polymers that provide a
wound healing in our body.[5] Normally, this has physicochemical and biological three-dimensional
been adopted in teeth with immature/open apices microenvironment for the cells to grow and
in which cellular responses are likely to be most differentiate, promoting cell adhesion and
favorable and can lead to root growth and apical migration.[11,12] Scaffolds can be natural (collagen,
closure. The ‘regenerated’ pulp tissue may not glycosaminoglycans), synthetic
closely resemble its physiological counterpart, (polycaprolactone, polyglycolic acid, polylactic
however. The use of triple antibiotic paste and acid) or mineral (hydroxyapatite, calcium
restorative materials that provide the best coronal phosphate).
seal in these revascularization procedures are Growth Factors
based on the contemporary methods to suppress Growth factors are extracellularly secreted
infectious/inflammatory events.[6] proteins/signals that bind to receptors on the cell
important. and signals morphogenesis /organogenesis during
TRILOGY OF REGENERATION epithelial-mesenchymal interactions. They
The concept of tissue engineering was introduced regulate the specialization of stem cells to the
by Charles Vacanti and Robert Langer (1993) as desirable cell type and mediates key cellular
an inter- disciplinary field that applies the events in tissue regeneration including cell
principles of engineering and life sciences proliferation, differentiation, chemotaxis and
towards the development of biological substitutes matrix synthesis.[13,14] Common growth factors are
that restore, maintain, or improve tissue bone morphogenic protein (BMP), fibroblast
function.[7] These approaches to repair and growth factior I or II (FGF), insulin like growth
regenerate depend on the basic principle of tissue factor I or II (IGF), colony stimulating growth
engineering; i.e. the interaction of a factor (CSF), epidermal growth factor (EGF),
scaffold/matrix with cells and signaling interleukin IL1-13, transforming growth factor-
molecules, albeit in the guise of natural wound alpha, beta (TGF α & β), vascular endothelial
healing events.[8] growth factor (VEGF), platelet derived growth
Stem Cells factor (PGDF) and nerve growth factor (NGF).
A stem cell is one that continuously divides and REGENERATIVE APPROACHES IN
produces progeny cells and can differentiate into ENDODONTICS
various other types of cells or tissues.[9] They are The various regenerative approaches tried over
categorized according to the lineage of these years have met with limited success. It
development as embryonic stem cells (foetal appears to be a promising option nevertheless, as
stem cells) and post-natal (adult) stem cells.[10] the techniques to achieve regeneration continue to
Oral stems cells come under the post natal stem improve. While revascularization is well
cell category and includes: documented and is easy to perform and less
 Dental pulp stem cells (DPSCs), expensive, other methods are still in various
 Stem cells of the apical papilla (SCAP), experimental stages.
 Inflammatory periapical progenitor cells Root Canal Revascularization
(iPAPCs), Revascularization of pulp can be defined as
 Periodontal ligament stem cells (PDLSCs), growth of undifferentiated stems cells of
 Stem cells from human exfoliated deciduous periapical region into the empty sterile/disinfected
teeth (SHED), root canal space.[15] Research on revascularization
 Dental follicle stem cells (DFSCs) commenced in the 1950’s and 60’s. Initially,
Scaffolds (Extra Cellular Matrix) researchers focused on methods to renovate the
The cells have to be seeded into an artificial ischemic pulp to vital pulp. The concept of guided
structure capable of supporting three dimensional tissue regeneration (GTR) was introduced by Dr.
tissue formations. In mid 1980’s, Dr Joseph Nygaard-Ostby (1961). Ostby and Hjortdal were
Vacanti and Robert Langer designed scaffolds the forerunners in the field of pulp
that were fabricated synthetically in laboratory revascularization whose first attempts in necrotic
tooth were unsuccessful.[16] Ostby demonstrated

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that periapical healing could be stimulated with appointment, use of ethylenediaminetetraacetic

the help of a blood clot inside the root canal.[17] acid (EDTA) as a final irrigant removed the
Johnson et al., in the 70’s and 80’s conducted smear layer, thereby releasing various growth
studies on dogs regarding the ability of pulp factors from dentin. In the same appointment,
tissue to regain blood supply by replanting delivery of stem cells into the root canal is
avulsed teeth and achieved revascularization in initiated by inducing bleeding,[24] and creating a
some teeth and resorption in others.[18,19] By early blood clot or placement of platelet rich plasma
2000, this procedure is considered as an which acts as scaffold for stem cells.[25,26] A
alternative to apexification. The concept of GTR leakage-free coronal seal is mandatory for the
was later customized by Dr. Trope with a success of the treatment. A biocompatible
modified clinical protocol (2004), which is still in material like mineral trioxide aggregate (MTA) or
current practice to accomplish revascularization. biodentine that provides a barrier over the blood
The procedure starts with access preparation and clot or PRP is used. The permanent coronal
disinfection of the root canal system. Irrigants are restoration is given to prevent bacterial
the primary agents that reduce the microbial load reinfection,[27] thereby achieving a triple seal. The
in infected root canal and include sodium revascularization procedure is schematically
hypochlorite (NaOCl) and chlorhexidine. NaOCl represented in Fig. 1.
in concentrations ranging from 0.5%-6% has been Postnatal stem cell therapy
used. Martin et al indicated that NaOCl in higher This method involves placement of postnatal stem
concentration had a detrimental effect on dentin cells into the disinfected root canal for
as well as on stem cells in the apical papilla.[20] regeneration of lost dental tissue. The first
Concentration of 1-1.5% had the least effect on postnatal stem cell therapy in medicine was done
stem cells.[20] Chlorhexidine is recommended for the treatment of severe combined immune
considering its wide antimicrobial activity and deficiency.[28] Postnatal stem cells may be sourced
substantivity. The interaction between the CHX from umbilical cord blood, peripheral blood, bone
and root dentinal surface provide effective and marrow, body fat, and other body tissues, like the
prolonged disinfection. The concomitant use of pulp tissue of teeth.[10] The drawbacks of postnatal
sodium hypochlorite and chlorhexidine should be stem cell therapy include low survival rates of
avoided as it leads to formation of injected cells, minimal chance of cells to migrate
parachloroaniline, a known carcinogen. Copious to different parts within the body,[29] which may
irrigation with saline is hence advised between lead to aberrant patterns of mineralization and
applications of these irrigants. A final rinse with difficulty in isolating the cells.
0.12% CHX in the first appointment gave better Pulp Implantation
disinfection (Happasalo).[22] Geisler however This concept seeks to renovate the two
recommended not using CHX or using it with dimensional cell cultures to a three-dimensional
caution, considering the cytotoxic effect of CHX structure by growing the cells in biodegradable
on stem cells.[22] Intracanal medicaments also membrane filters.[14] Bohl et al.,[28] reported the
have a role in the disinfection protocol. Calcium formation of tissue with high cell density similar
hydroxide was used in the earlier attempts to the natural pulp when pulp cells are cultured in
followed by use of antibiotic pastes. Triple vitro on polyglycolic acid (PGA). Buurma et
/double antibiotic paste is now being used at al.,[30] observed extracellular matrix to be
0.1mg/ml concentration. The combination expressed in immunocompromised mice when
consists of ciprofloxacin, metronidazole, and PGA was seeded with pulp cells and implanted
minocycline in 1:1:1 ratio, applied for 21 days. into subcutaneous spaces. Angiogenesis occurred
Since minocycline was observed to cause through PGA implants after three weeks of
discoloration to the crown, alternatives like implantation. The pulp stem cells must be
cefaclor, amoxicillin, cefroxadin, fosfomycin, and organized in such a way that it should support cell
rokitamycin have been mentioned by various organization and vascularization. Growing dental
authors.[8] Discoloration can be prevented by pulp cells on collagen matrix was not successful
sealing the dentinal tubules with dentin bonding and matrices like vitronectin and laminin require
agent[23] before antibiotic placement. In the final further investigation.[31] The advantage of this

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Fig. 1: Revascularisation procedure

philosophy is the ease of growing cells on filters Injectable Scaffold Delivery
within the laboratory and their existence as Injectable scaffolds such as polymer hydrogels
collective sheets which are a more stable than can be delivered by a syringe. These scaffolds
unconnected cells and can be easily injected into provide an excellent substrate and can support
empty disinfected root canal systems. However, cell proliferation and provide nutrition to the
there is a need for specialized procedures to cells. The scaffolds are soft and three dimensional
ensure proper adherence of cells to root canal in nature and can be easily placed inside the root
walls and handling fragile filters. canal. These are non invasive and easy to handle.
Scaffold Implantation The success of these scaffolds is yet to be studied
Pulp stem cells may be ordered into a three in clinical settings.[14]
dimensional arrangement that can sustain cell Three dimensional cell printing
organization and vascularization. This can be A rapid prototype of computer-aided 3D
consummate using a porous polymer scaffold technology in which cells are dispensed layer by
which is seeded with pulp stem cells. A scaffold layer using an ink-jet like gadget in a 3D
should contain growth factors that aid hydrogel.[14,33] This technique facilitates precise
proliferation and differentiation of stem cells, positioning of stem cells,thereby creating tissues
leading to enhanced and more rapid tissue that mimic the natural tooth pulp. Ideal
development. The scaffolds that are used for this positioning of cells should be in such a way that
procedure are either synthetic or natural. placing odontoblastoid cells around the periphery
Synthetic scaffolds include polyester materials to sustain and repair dentin, with fibroblasts
like polycaprolactone (PCL), polyglycolic acid arranged in the core of pulp supporting vascular
(PGA) and polylactic acid (PLA). Natural and nerve cells. The shortcoming of using the 3D
scaffolds may be collagen, chitosan, fibrin or cell printing technique is difficulty in symmetrical
glycosamino glycans. Dentin chips have been spatial arrangement of pulp tissue in an
reported to stimulate reparative dentin bridge asymmetric apico-coronal portion of root canal.
formation in teeth with exposed pulp.[32] Dentin Gene Therapy
chips will be a reservoir of growth factors thereby Gene therapy is a relatively new field in
acting as a matrix for pulp stem cell regenerative endodontics where preliminary
attachment.[14] The scaffold may also contain attempts focuses on delivering genes that are
nutrients, promoting cell survival and growth. To capable of matrix formation and mineralization
achieve the goal of pulp tissue renovation, into pulp space to promote tissue repair using
scaffolds must meet some definite requirements – vectors. Its practicality, potential health hazards
biocompatibility, biodegradability and high and ethical concerns are much demanding, which
porosity.[14] accounts to the limited exploration of the

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technique in endodontics. Once successful, this difficult to remove from the root canal and causes
technique could be a landmark development in discoloration of the crown portion over a period
the field of regenerative endodontics.[12] of time. Calcium hydroxide has deleterious effect
Bioengineered tooth like weakening the root dentin,[21] with a potential
The current philosophy in tissue engineering is for fracture.
developing/regenerating a whole new organ. This Removal of smear layer
concept can be made possible by two approaches Even though there is minimal instrumentation of
- seeding the tooth germ cells in a biodegradable the root canal in regenerative procedure, there is
tooth shaped scaffold or recreating the tooth germ limited smear layer formation. This prevents stem
from detached mesenchymal and epithelial cells from adhering on to root canal walls,
cells.[34] In the first method, a mixture of cells is contributing to failure of treatment. A 17%
introduced into a scaffold of desired shape. The solution of EDTA as final rinse in the root canal
scaffolds used are made of polyglycolate (PGA) will aid in removal of smear layer as well as
and poly-L-lactate-co-glycolate (PLGA) and enhance the attachment of the stem cells by
collagen sponge are preferred. In the other providing a better surface on the root dentin for
method, multi-cellular assembly and self- their survival. Other chemicals used for this
reorganization of each mesenchymal and purpose are doxycycline, citric acid and MTAD.
epithelial cell is achieved first. Then, reciprocal Developing an ordered functional pulp tissue
interactions between epithelial and mesenchymal Developing a functional tissue is still a problem
cell layers initiate organogenesis that regulates in regeneration. Despite best efforts to recreate
differentiation and morphogenesis. lost tissue, the type of tissue is still unknown. The
CHALLENGES IN REGENERATIVE development of the normal harmony and spatial
ENDODONTICS arrangement of dentin pulp complex is still in
 Disinfection of root canal research. The 3D cell printing will be a possible
 Removal of smear layer solution in the near future, but the problem with
 Developing an ordered functional pulp this technology is the difficulty in exact
tissue placement and positioning inside root canal. Only
 Appropriate coronal seal by achieving such a balanced spatial oriented cell
 Appropriate method to measure the lineage in the root canal through regeneration can
clinical outcome this method be deemed successful.
Disinfection of root canal Appropriate coronal seal
One of the biggest challenges encountered in A leakage-free coronal restoration is mandatory
endodontics is inadequate disinfection of infected in regenerative endodontics as well as in
root canals. Reduction of microbial level is subsequent restorative procedures. The material
crucial for the success in both regenerative that provides such seal should be biocompatible,
endodontics as well as routine endodontic have to maintain cell viability of the regenerated
procedures. Since instrumentation in regenerative pulp, should minimize microleakage and provide
procedure should be minimal, disinfection by adequate adhesion to the overlaying restorative
chemical means is recommended in the form of material.[36] The materials under consideration are
irrigants and medicaments. NaOCl is the most calcium silicate based cements like mineral
widely recommended irrigant (concentration ≤ trioxide aggregate (MTA) and biodentine.
1.5%) . 17 % EDTA is another chemical agent Appropriate method to measuring the clinical
that is recommended in regeneration procedure outcome
as it helps in the dissolution and release of Ethical issues prevent histological analysis of the
impregnated growth factors in root dentin during implanted regenerative cells. Noninvasive tests
dentinogenesis.[35] These growth factors enhance such as laser doppler blood flowmetry, pulse
the stem cell survival and its proliferation. EDTA oximetry and lack of clinical signs or symptoms
is also useful in reversing the detrimental effect of and radiographic signs like completion of root
high concentration of NaOCl. Regarding formation and disappearance of periapical lesions
intracanal medicaments, triple antibiotic paste is have to be relied upon to verify vitality of the
pulp. Pulp sensibility tests like thermal tests and

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electric pulp test are not reliable in checking the BIBLIOGRAPHY

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