Regenerative endodontics

REGENERATIVE
ENDODONTICS
DR. VIPUL ARORA
MDS 3RD YEAR
KD DENTAL COLLEGE

INTRODUCTION
 The high susceptibility of teeth to damage ,combined with the
non regenerative nature of dental tissues , emphasizes the need
for replacement tooth therapies .
 Until the present time the field of restorative dentistry and
material sciences have combined efforts to produce a variety of
synthetic materials for use in restoration of damaged dental hard
tissues .
 Although these materials and therapies have proved effective ,
they do not exhibit the same mechanical and physical properties
as naturally formed enamel and dentin .

SHIFT IN PARADIGM
FROM REPLACEMENT THERAPIES
TO REGENERATIVE THERAPIES

• Recent advances in biotechnology have made possible exciting
advancements in the field of stem cells and regenerative medicine.
• Opportunities now exist for maintaining or trying to regain pulp
vitality, attempts to induce dentinogenesis and to revascularize an
infected root canal.
• This presentation is an attempt to explain the various aspects of
regenerative endodontic procedures and its role as an alternative to
classical endodontic treatment where the whole pulp is removed
and replaced with inert materials.
Regenerative Endodontics : A review of current status and a call for action. Peter E Murray, Franklin Garica-
Godoy, Kenneth M Hargreaves. J Endod, 33(4) April 2007

• Regenerative dental procedures originated in 1952, when B.W. Hermann
reported on the application of calcium hydroxide in a case report of vital pulp
amputation.
• Seltzer et al (1958) studied pulpal repair in Dog teeth and concluded that
Ca(OH)2 was the only calcium salt capable of stimulating the pulp to lay
down reparative dentin.
• Rule and Winter (1966) opened canals without the use of a local anaesthetic
and cleaned to the level of bleeding tissue over which polyantibiotic paste
was placed. A periapical radiograph taken 3 years later showed completed
root formation.
• Myers et al (1974) in their study hypothetized that when the canals of the
teeth with periapical infection were filled with whole blood and/or blood
substitutes, dental pulp could regenerate.
Regenerative endodontics: A state of the art, journal of dental research,2011

• "Tissue engineering" was coined by Langer and Vacanti in 1993 to describe
the process by which the tissues and organs are generated by cell
transplantation with or without a scaffold.
• Gronthos et al (2000) first isolated dental pulp stem cells (DPSCs). These cells
could regenerate dentin-pulp complex composed of a mineralized matrix of
tubules lined with odontoblasts and fibrous tissue.
• Iwaya et al (2001) reported a case of a 13 year old patient with an immature
necrotic mandibular second premolar treated with double antibiotic paste
and inducing bleeding in the canal. Root end formation was seen in 5 months
and thickening was seen in 30 months.
• Banchs et al (2004) reported a case of an immature permanent tooth with
apical periodontitis in a 11 year old boy who was treated with triple antibiotic
paste and mechanical irritation of the apex. Root formation was complete in
2 years.
Regenerative endodontics: A state of the art, journal of dental research,2011

DEFINITIONS
• REGENERATION refers to a type of
healing in which new growth
completely restores portions of
damaged tissue to their normal state.
• REPLACEMENT refers to a type of
healing in which severely damaged or
non-regenerable tissues are repaired by
the laying down of connective tissue, a
process commonly referred to as
scarring.
Tissue Repair: The Hidden Drama. Kristine Krafts. Organogenesis. 2010 Oct-Dec; 6(4): 225–233.

• TISSUE ENGINEERING-
Langer and Vacanti (1993)-
An interdisciplinary field that applies the principles of engineering
and life sciences toward the development of biological substitues
that restore, maintain or improve tissue or organ function

REGENERATIVE ENDODONTICS
• Regenerative endodontic procedures
are biologically based procedures
designed to replace damaged
structures, including dentin and root
structures as well as cells of the
dentin pulp complex.

• REVASCULARIZATION is defined as re-
establishment of vascular supply to
immature permanent teeth.
• REVITALIZATION describes ingrowth of
vital tissue that does not resemble the
original lost tissue.
Law AS. Outcomes of Regenerative Endodontic Procedures. Dent Clin N Am. 2012;56:627–637.

• TROPE claimed that the term revascularization was chosen because
the nature of the tissue formed post-treatment was unpredictable,
and the only certainty was the presence of a blood supply; hence it
was ‘‘revascularized.’’
• HUANG AND LIN challenged the term revascularization as applied to
endodontic procedures and believed it was more applicable to events
that followed dental trauma. They suggested the term induced or
guided tissue generation and regeneration.
• More recently, Lenzi and Trope suggested the term revitalization as
being more appropriate because it is descriptive of the nonspecific
vital tissue that forms in the root canal.
2 Huang GT, Lin LM. Letter to the editor: comments on the use of the term ‘‘revascularization’’ to
describe root regeneration. J Endod 2008;34:511. author reply 511–2.

OBJECTIVES OF REGENERATION
Regenerate pulp-like tissue, ideally the pulpo-dentin complex.
Regenerate damaged coronal dentin, such as following a carious
exposure.
Regenerate resorbed root, cervical or apical dentin.
Peter E Murray, Franklin Garcia-Godoy. Stem cells and regeneration of the pulpodentin complex. In: Kenneth M Hargreaves, Harold E
Goodis, Franklin R Tay, Seltzer and Bender’s Dental Pulp, 2nd Ed, 2012. Quintessence Publishing Co Inc.

Why the need for Regenerative Endodontics?
Peter E Murray, Franklin Garcia-Godoy. Stem cells and regeneration of the pulpodentin complex. In: Kenneth M Hargreaves, Harold E Goodis, Franklin R Tay,
Seltzer and Bender’s Dental Pulp, 2nd Ed, 2012. Quintessence Publishing Co Inc

VITAL TEETH V/S ROOT CANAL TREATED TEETH
• Teeth with vital pulps are much more resistant to bacterial invasion
into the dentinal tubules than teeth with root fillings.
• In teeth with pulps, the dentinal tubules are occupied with
odontoblastic processes and dentinal fluid, which may behave
collectively as a positively charged hydrogel that is capable of
arresting a great number of bacteria that enter the pulp.
An overview of the dental pulp: its functions and responses to injury. C. Yu, PV Abbot. Australian Dent J, Endodontic supplement
2007;52:1

• The outward flow of dentinal fluid is important in the pulp’s defence
against the entry of harmful substances.
• The pulp’s cells, the odontoblasts and undifferentiated mesenchymal
cells (Which may differentiate into dentine-forming cells if stimulated)
retain the ability to form dentine throughout life.
An overview of the dental pulp: its functions and responses to injury. C. Yu, PV Abbot. Australian Dent J, Endodontic supplement
2007;52:1

TISSUE ENGINEERING TRIAD
GROWTH
FACTORS
STEM CELLS
SCAFFOLD
Regenerative
Endodontics: A Review
of Current Status and a
Call for Action. Peter E
Murray, Franklin Garcia-
Godoy, Kenneth M
Hargreaves. J Endod
33(4), April 2007, 377-90

MAJOR DOMAINS OF REGENERATIVE ENDODONTIC
PROCEDURES
Stem Cells
Growth
Factors
Tissue
engineering
materials
Cell, tissue
and organ
culture
Regenerative
Endodontics
Regenerative Endodontics: A Review of Current Status and a Call for Action. Peter E Murray, Franklin Garcia-Godoy, Kenneth M Hargreaves. J
Endod 33(4), April 2007, 377-90

STEM CELLS
• Stem cells are primitive cells that can
differentiate and regenerate organs in
different parts of the body such as
heart, bones, muscles and nervous
system.
• A stem cell is defined as a cell that has
the ability to continuously divide and
produce progeny cells that differentiate
into various other types of cells and
tissues.
Imperative Role of Dental Pulp Stem Cells in Regenerative Therapies: A Systematic Review
Niger J Surg. 2014 Jan-Jun; 20(1): 1–8.doi: 10.4103/1117-6806.127092

• The term stem cell was proposed for scientific use by Russian
histologist Alexander Maksimov in 1909.
• He was the first to suggest the existence of hematopoietic stem cells
(HSC) with the morphological appearance of a lymphocyte, capable of
migrating throughout the blood to micro ecological niches that would
allow them to proliferate and differentiate.

• The hallmark features of stem cells are
1)Self renewal
2)Plasticity/Pluripotentiality

Types of Stem cells and potency
Stem cell
type
Cell Plasticity Source of stem
cell
Totipotent Each cell can develop into
a new individual
Cells from early (1-3
days) embryos
Pluripotent Each cell can form any
(over 200) cell types
Some cells of
Blastocyst (5-14 days)
Multipotent Cells differentiated but can
form a number of other
tissues
Fetal tissue, cord blood
and postnatal stem
cells including Dental
pulp stem cells.
Regenerative Endodontics : A review of current status and
a call for action. Peter E Murray, Franklin Garica-Godoy,
Kenneth M Hargreaves. J Endod, 33(4) April 2007

Sources of stemcells
Tuch BE (2006). "Stem cells—a clinical update". Australian Family Physician 35 (9): 719 21. PMID 16969445

Dental Stem cells
At least five different types of postnatal mesenchymal stem cells have
been reported to differentiate into odontoblast-like cells, including
Stem cells of apical papilla [SCAP]
Inflammatory periapical progenitor cells [iPAPCs]
Dental pulp stem cells [DPSCs]
Periodontal ligament stem cells [PDLSCs]
Bone marrow stem cells [BMSCs]

Dental follicle stem cells [DFSCs]
Tooth germ progenitor cells [TGPCs]
Salivary gland stem cells [SGSCs]
Stem cells from human exfoliated deciduous teeth [SHED]
Oral epithelial stem cells [OESCs]
Gingival derived mesenchymal stem cells [GMSCs]
Periosteal derived stem cells [ PSCs]

DENTAL PULP STEMCELLS
{DPSC}
• DPSCs were first isolated from
human permanent third molars
in 2000.
• They were characterized as
clonogenic and highly
proliferative.

• Dentin and Pulp-like tissue were
generated following the
transplantation of DPSCs in
hydroxyapatite/ tricalcium
phosphate scaffolds into
immunodeficient mice.
• It is now recognized that DPSCs can
also differentiate into osteoblasts,
chondrocytes and myoblast-like cells
and demonstrate axon guidance.
Imperative Role of Dental Pulp Stem Cells in Regenerative Therapies: A Systematic Review Niger J
Surg. 2014 Jan-Jun; 20(1): 1–8.doi: 10.4103/1117-6806.127092

• Sometimes pulp stem cells are called odontoblastoid cells, because
these cells appear to synthesize and secrete dentin matrix like the
odontoblast cells they replace.
• Carnevale et al. have recently found that human amniotic fluid stem
cells and human DPSCs differentiate into insulin-producing cells,
offering a non-pancreatic, low-invasive source of cells for islet
regeneration.

ROLE OF DPSC
• The role of DPSCs in successful reconstruction
of cornea has also been explored in the recent
past.
• In an animal study by Gomes et al., a tissue
engineered DPSC sheet was transplanted on
the corneal bed and then covered with de-
epithelialized human amniotic membrane.
Histological analysis at three months
postoperative phase confirmed that healthy
uniform corneal epithelium was formed.
Moreover, it was concluded that tissue
engineered DPSC sheet was successful for the
reconstruction of corneal epithelium.
Imperative Role of Dental Pulp
Stem Cells in Regenerative
Therapies: A Systematic Review
Niger J Surg. 2014 Jan-Jun; 20(1): 1–
8.doi: 10.4103/1117-6806.127092

• Regardless of the recent advances in prevention and
treatment of myocardial infarction (MI), it remains one of
the major causes of mortality worldwide.
• In this perspective, cardiomyocytic differentiation of
DPSCs has been studied by various researchers.
• The overall capability of the stem cells derived from the
bone marrow stem cells (BMSCs), adipose tissue cells
(ATSCs) and DPSCs to differentiate to cells with a cardiac
phenotype was first estimated by Arninam and co-
workers.
• They postulated that the tissue specific mesenchymal
stem cells (MSCs) can change into cardiomyocytes and
support the potential use of MSCs in the stem cell based
cardiac therapies. Furthermore, the therapeutic potential
of DPSCs in repair of myocardial infarction was evaluated
by Gandia et al. in 2008, who concluded that human
DPSC secrete multiple pro-angiogenic apoptotic factors.
Imperative Role of Dental Pulp Stem
Cells in Regenerative Therapies: A
Systematic Review Niger J Surg. 2014
Jan-Jun; 20(1): 1–8.doi: 10.4103/1117-
6806.127092

Treatment of infertility employing DPSCs
• The prospective of DPSCs can also be used in the
treatment of infertility.
• Kerkis et al. in a study
successfully segregated HDPSCs and
injected them to the testis of live male mice. The
mice were killed at various intervals after the
injection and their testis were examined to see
whether stem cells survived. It was postulated that
the stem cells settled in testes and also
differentiated into cells that were producing viable
sperm
Imperative Role of Dental
Pulp Stem Cells in
Regenerative Therapies: A
Systematic Review Niger J
Surg. 2014 Jan-Jun; 20(1): 1–
8.doi: 10.4103/1117-
6806.127092

stem cells from human exfoliated deciduous teeth (SHED)
• SHED cells are highly proliferative stem cells
from exfoliated deciduous teeth.
• They are capable of differentiating into a
variety of cell types including osteoblasts,
neural cells, adipocytes and odontoblasts.
• They can also induce dentin and bone
formation.
• SHED cells have a higher proliferation rate
than DPSCs, suggesting they represent a more
immature population of multipotent stem
cells.
Stem Cells from Human Exfoliated Deciduous Teeth- A Boon to Dentistry International Journal of Oral Health and Medical
Research | ISSN 2395-7387 | JULY-AUGUST 2016 | VOL 3 | ISSUE 2

STEM CELLS OF APICAL PAPPILA
{SCAP}
• SCAP cells are found in the apical papilla
located at the apices of developing teeth
at the junction of the apical papilla and
the dental pulp.
• The apical papilla is essential for root
development.
• SCAP cells were first isolated in human
root apical papilla collected from extracted
third molars.
The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot

The Potential Role of SCAP in Pulp Healing and Regeneration Immature Teeth with
Periradicular Periodontitis or Abscess UNdergo Apexogenesis

DENTAL FOLLICLE STEM CELL
{DFSC}
• The dental follicle is a homogeneous layer
of ectomesenchymal cells surrounding the
tooth germ outside of the outer dental
epithelium and dental papilla in early
stages of tooth bud formation.
• One important biological function of the
dental follicle is the coordination of teeth
eruption.
Dental follicle stem cells and tissue engineering journal of Oral Science, Vol. 52, No. 4, 541-552, 2010

• The dental follicle harbors progenitor cells for
the periodontium, the supporting apparatus
of the tooth.
• Recently, progenitor cells were identiﬁed in
dental follicles of bovine tooth germs
released by digestion with bacterial
collagenase.
Dental follicle stem cells and tissue engineering journal of Oral Science, Vol. 52, No. 4, 541-552, 2010

Bone marrow mesenchymal stem cells
{bmmsc}
• Mesenchyme is embryonic connective tissue that is derived from
the mesoderm and that differentiates into hematopoietic and connective
tissue
• Mesenchymal stem cells, or MSCs, are multipotent stromal cells that
can differentiate into a variety of cell types,including: osteoblasts (bone
cells), chondrocytes (cartilage cells), and adipocytes (fat cells).
• Adipose tissue and bone marrow are rich sources of MSCs
• When compared to bone marrow, there is more than 500 times more stem
cells in 1 gram of fat when compared to 1 gram of aspirated bone marrow
Nardi, N. Beyer; da Silva Meirelles, L. (2006). "Mesenchymal Stem Cells: Isolation, In Vitro Expansion and Characterization". In Wobus, Anna M.; Boheler,
Kenneth. Stem Cells. Handbook of experimental pharmacology 174. pp. 249–82

Stem cells banking
STEMADE
 India’s first and largest Dental Stem cell Bank.
 In Mumbai, Delhi, Chennai, Chandigarh, Ludhiana, Ambala and
Nasik.
 Stem cells are collected from younger patients [<3O years] of age.

Storage of stem cells
• CRYOPRESERVATION(cryo – cold)
• It is considered to be a very effective method for
maintaining the viability of the transported stem
cells by cooling to subzero temperatures, typically –
196°C (neelampari) where the biological activity
concludes leading to cell death.
• Procedure involves slow cooling at 1 to 2 °C/min in
the presence of a cryoprotectant, Dimethyl
SulphOxide (DMSO) to avoid the damaging effects of
intracellular ice formation is considered standard.1

MAGNETIC FREEZING:
In this method, a weak magnetic field is
applied to the water and tissues thereby
lowering the freezing point of body up to
6-7° C. It ensures distributed low
temperature without the cell wall damage
resulted by ice expansion and nutrient
drainage due to capillary action, as caused
by conventional freezing methods.
 Magnetic freezing is considered to be
more reliable as well as relatively cheaper
as compared with cryopreservation.
Stem Cells from Human Exfoliated
Deciduous Teeth- A Boon to Dentistry
International Journal of Oral Health and
Medical Research | ISSN 2395-7387 |
JULY-AUGUST 2016 | VOL 3 | ISSUE 2

GROWTH FACTORS
Growth factors are proteins that bind to receptors on cells and induce
cellular proliferation and/or differentiation.
Growth factors are polypeptides or proteins that bind to specific
receptors on the surface of target cells

• They can initiate a cascade of intracellular signaling and act in either
an autocrine or paracrine manner.
• Various growth factors and their receptors have been shown to be
present at the enamel organ-dental papilla interface by
immunohistochemistry and in situ hybridization.
• Dentine is considered as a reservoir of growth factors [ TGF˗ß1, FGF,
VEGF, BMP].
• Their release from the matrix appears to hold the key to the pulp
healing process
Roberts-Clark DJ, Smith AJ. Angiogenic Growth Factors in Human Dentine Matrix. Arch
Oral Biol 2007;45: 1013-16.

• Demineralization of dentin can lead to release of growth factors
following application of cavity etching agents or restorative materials
and even caries.
• Indeed, the therapeutic action of Ca(OH)2 and MTA may be due to
their extraction of growth factors from the dentin matrix.

• Transforming growth factor-𝛽 (TGF-𝛽)
Stimulates proliferation of osteoblasts
Synthesis of collagen and fibronectin
Stimulates angiogenesis
• Platelet-derived growth factor (PDGF)
Angiogenic effect on endothelial cells
• Vascular endothelial growth factor (VEGF)
Initiates angiogenesis
Dental Stem/Progenitor cells. Sahng G. Kim et al. Dent Clin N Am 56 (July 2012), Elsevier Inc.

• Insulin growth factor-1 (IGF-1)
Stimulates osteoblast proliferation
• Fibroblast growth factor (FGF)
Stimulates osteoblast proliferation
Chemotactic effects towards human osteoblasts
• Epidermal growth factor (EGF)
Stimulation of cell proliferation and extracellular matrix
Chemotactic effect on periodontal fibroblast cells
Dental Stem/Progenitor cells. Sahng G. Kim et al. Dent Clin N Am 56 (July 2012), Elsevier
Inc.

SCAFFOLDS
• In tissue engineering, the selection of a suitable scaffold is critical.
• Scaffolds can be identified as biocompatible structures that support cells
growth and provide a suitable environment for tissue formation.
• Scaffolds are three-dimensional structures that provide an initial
framework for the cells and can be used to deliver morphogenic
molecules.
• Good scaffolds should allow cell attachment, proliferation, migration,
differentiation, and provide mechanical support for the extracellular matrix
generation
Shehab El-Din M. Saber. Tissue engineering in endodontics. Journal of Oral Science 2009;51:495-507.

Requirements of scaffolds
• It should provide support for delivering cells and/or growth factors to the
proposed sites of tissue regeneration.
• It should reflect the microenvironment of target tissues/organs to facilitate
cell growth and ultimately integration to the host.
• It should be porous to allow placement of cells and growth factors.
• It should allow effective transport of nutrients, oxygen, and waste.
• It should be biodegradable, leaving no toxic byproducts.
• It should be replaced by regenerative tissue while retaining the shape and
form of the final tissue structure.
• It should be biocompatible.
• It should have adequate physical and mechanical strength.
Shehab El-Din M. Saber. Tissue engineering in endodontics. Journal of Oral Science 2009;51:495-507.

ROOT CANAL REVASCULARIZATION VIA BLOOD
CLOTTING

ADVANTAGES:
• Technically simple
• Avoids the possibility of immune rejection
DISADVANTAGE
Concentration and compositions of cells trapped in the fibrin clot is
unpredictable.

POSTNATAL STEMCELL THERAPY
 They can be derived from skin, buccal mucosa, fat or bone.
 One of the obstacle is identification of the stem cell source capable
of differentiating into cell population found in adult pulp.
They are easy to harvest, but the cells can migrate to different
locations. Thus need to hold it together with a fibrin clot or other
scaffold material.

PULP IMPLANTATION
Stem cells are grown on biodegradable membrane filters.
These filters are implanted into disinfected root canal systems.
ADVANTAGE:
Cells are relatively easy to grow on filters in laboratory.
Filters localizes the postnatal stem cells in root canal system.
DISADVANTAGE
Filters are very thin layers of cells, they are fragile and thus difficult to
place them in root canal without breakage.

SCAFFOLD IMPLANTATION
3D structure which supports the cell organization and vascularity.
Accomplished using porous polymer scaffold seeded with pulp stem
cells, along with growth factors.
It should be biodegradable, and
porous in nature3D structure which
supports the cell organization and vascularity.

INJECTABLE SCAFFOLD DELIVERY
• HYDROGELS are injectable scaffold that can be delivered by syringe.
• Easy delivery in the root canal space.
• They may promote pulp regeneration by causing the cell differentiation and
proliferation but the problem includes its limited control over tissue
formation and development.
• It is still at an early stage of research, and has yet to be proven to be
functional.

3D CELL PRINTING
• Ink jet like device is used to dispense layers of cells suspended in a
hydrogel to create the structure of the tooth pulp.
• It constructs the tissue that mimic the natural tooth pulp tissue structure.
• It can be used to precisely position cells odontoblastoid cells around the
periphery to maintain and repair dentin, with fibroblasts in the pulp core
supporting a network of vascular and nerve cells.
• The main disadvantage is that careful orientation of the pulp tissue is
required during its placement into root canal systems.
• Research has yet to show that it can create functional tissue in vivo
3D bioprinting for biomedical devices and tissue engineering: A review of recent trends and advances

GENE THERAPY
• Gene therapy is recently used as a
means of delivering genes for growth
factors, morphogens, transcription
factors, extracellular matrix molecules
locally to somatic cells of individuals
with a resulting therapeutic effect.
• The gene can stimulate or induce a
natural biological process by
expressing a molecules involved in
regenerative response for the tissue
of interest.
Bonadio J, Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue
regeneration. Nat Med 1999; 5:753–9.

Use of GENE delivery in endodontics would be to deliver
mineralizing genes into pulp tissue to promote the tissue
mineralization.
It is a relatively new field and evidence is lacking to demonstrate that
this therapy has the potential to rescue necrotic pulp.
Bonadio J, Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue
regeneration. Nat Med 1999; 5:753–9.

Majority of currently published regenerative endodontic procedures
involve the evoked bleeding and the formation of blood clot to act as a
scaffold.
But blood clot is often difficult to achieve as it does not have
controllable biodegradation, incorporation of growth factors, it also
contains a great number of hematopoietic cells which after death release
their enzymes which may be detrimental to the stem cell survival.
ANOTHER APPROACH IS THE USE OF PLATELET RICH
PLASMA/ PLATELET RICH FIBRIN.

PLATELET RICH PLASMA
{PRP}
The Platelet Rich Plasma (PRP), also termed autologous platelet gel
(Autologous Platelet Gel), is a gel at high concentration of autologous
platelets suspended in a small amount of plasma after centrifugation of
the blood of the patient.
The platelets in fact play a fundamental role in mediating the healing of
damaged tissue due to its ability to release growth factors, including
PDGF, TGFβ, VEGF, IGF-1, FGF, and EGF.
Comparison between PRP , PRGF and PRF: lights and shadows in three similar but different protocol
European Review for Medical and Pharmacological Science2015;19:927-930

• The granules contained in platelets are also a source of cytokines,
chemokines and many other proteins variously involved in
stimulating proliferation and cellular maturation, in modulating
inflammation and activate other cells by regulating tissue
homeostasis and regenerative processes2

METHOD TO OBTAINPRP
• Venous blood is drawn into a tube containing an anticoagulant to avoid platelet
activation.
• "SOFT SPIN” which allows blood separation into three layers {22OO rpm for 5
min}
bottom-most RBC layer
Topmost acellular plasma layer called PPP [Platelet poor plasma]
an intermediate PRP layer called the "buffy coat".
• Using a sterile syringe, the operator transfers PPP, PRP and some RBCs into
another tube.

• HARD SPIN” - a second centrifugation.. [32OOrpm for 4 min]
• This allows the platelets (PRP) to settle at the bottom of the tube.
• PPP, found at the top, is removed with a syringe and discarded, and
the remaining PRP is taken.

PLATELET RICHFIBRIN
• The Platelet Rich Fibrin (PRF) is a quite modern platelet concentrate,
it is achieved with a simplified preparation, with no biochemical
manipulation of blood.
• Blood is collected in tubes without an anticoagulant and immediately
centrifuged at 2700 RPMfor 12 mins.
• Because there is no anticoagulant the blood starts coagulating as soon
as it comes in contact with the glass tube.

This technique does not require
anticoagulants or bovine thrombin (or any
other gelling agent)This feature make this
product easily usable, with a low rate of
mistakes during the preparation stage.
• The 3 layers found are :
Top most PPP
Middle PRF clot
Bottom RBCs

• The PRF has been studied by many research groups, and it has been
shown that it is able to stimulate osteogenesis in bone environment,
in addition to angiogenesis; Furthermore, it provides a scaffold
consisting of fibrin that allows cellular migration.

RECENT ADVANCEMENT IN PRF
• Recently, Some investigators have formulated a new protocol for PRF
where centrifugation procedures have been altered to further improve
tissue regeneration.
• While standard PRF is centrifuged at 2700 rpm for 12 min, the advanced
platelet-rich fibrin (A-PRF) is centrifuged at slower speeds (1500 rpm, 14
min).
• This modification to centrifugation protocol has previously been shown to
increase platelet cell numbers and monocytes/macrophages behavior.
Comparative release of growth factors from PRP, PRF, and advanced-PRF SPRINGER JAN 2016

INJECTABLE PRF
• Centrifuged at 700 rpm for 3 min.
• Increase platelet cell numbers and monocytes/macrophages behavior.

GOALS OF REGENERATIVE ENDODONTIC
PROCEDURES
• The primary goals are the elimination of symptoms and the evidence
of bony healing.
• Secondary goals (which are desirable but perhaps not essential)
include increased root wall thickness and/or increased root length.
• A tertiary goal (which, if achieved, indicates a high level of success) is
a positive response to vitality testing.
Law AS. Outcomes of Regenerative Endodontic Procedures. Dent Clin N Am. 2012;56:627–637.

CLINICAL PROTOCOL FOR REGENRATIVE ENDODONTIC
PROCEDURES
AAE Protocol

AAE PROTOCOL
• Case selection and informed consent
• Access and disinfection
• Stimulates scaffolds and seal
• Follow ups

• CASE SELECTION :
Tooth with necrotic pulp and an immature apex
Pulp space not needed for post/core, final restoration
No known allergies to antibiotics if intended for use
Compliant patient (parent/guardian)
• INFORMED CONSENT :
Two (or more) appointments
Use of antimicrobial(s)
Possible adverse effects: staining of crown/root, lack of response to
treatment, pain/infection.
Alternatives: MTA apexification, no treatment, extraction (when deemed
non-salvageable)

First appointment- Access and Disinfection
• Local anesthesia, rubber dam isolation,
access.
• Copious, gentle irrigation with 20ml 1.5%
NaOCl.
• Dry canals
• Place triple antibiotic paste or calcium
hydroxide.

• Consider sealing pulp chamber with a dentin bonding agent [to minimize
risk of staining]
• mix 1:1:1 ciprofloxacin:metronidazole:minocycline and deliver into canal
system.
• If triple antibiotic is used, ensure that it remains below CEJ (minimize
crown staining). As an alternative, Ca(OH)2 does not cause staining.
• Seal with 3-4mm Cavit, followed by IRM, glass ionomer cement or another
temporary material.
• Dismiss patient for 3-4 weeks

Second appointment – Stimulate, Scaffold and Seal
• Assess response to initial treatment. If there are signs/symptoms of
persistent infection, consider additional treatment with the antimicrobial,
or an alternative antimicrobial. Recall the patient in about 3-4 weeks as
before.
• Anesthesia with 3% mepivacaine without vasoconstrictor, rubber dam,
isolation
• Copious, slow irrigation with 20ml 17% EDTA, followed by normal saline.
• Dry with paper points

• Create bleeding into canal system by over-
instrumenting Stop bleeding at a level that allows for
3-4 mm of restorative material.
• Place CollaPlug /Collacote over clot.
• Place 3-4mm of MTA and reinforced glass ionomer and
place permanent restoration. Glass ionomer may be an
alternative to MTA in cases where discoloration of the
crown is a potential concern.

Follow up – AAE protocol
Clinical and radiographic examination:
• No pain or soft tissue swelling (often observed between first and second
appointments)
• Resolution of apical radiolucency (often observed 6–12 months after treatment)
• Increased width of root walls (this is generally observed before apparent increase in
root length and often occurs 12–24 months after treatment)
• Increased root length.
• Pulp vitality test

HURDLES TO OVERCOME
• The non-dental stem cells should also be explored for dental
applications.
• Appropriate vascularized scaffolds are required that are biodegradable
and have the same rate of degradation as rate of formation of
engineered tissue construct.
• Distribution of cells in scaffolds.
• A better understanding and control over growth factors to get the
desired quality tissue construct is required

CONCLUSION
• Regenerative endodontic strategies have tremendous potential to
be an effective, safe, and biological mode to save teeth.
• Considerable research and development efforts are required to
advance the regenerative therapeutics to next level.
• With new discoveries, innovative ideas, and high-quality research,
in the future, the scope of regenerative endodontics might increase
to include the replacement of periapical tissues, gingiva, and even
whole teeth.

REFERENCES
COHEN’S PATHWAYS OF THE PULP]
 REGENERATIVE ENDODONTICS: A REVIEW OF CURRENT STATUS AND
A CALL FOR ACTION, PETER E. MURRAAY ET AL, JOE 2007]
PLATELET RICH FIBRIN AS A BIOFUEL FOR TISSUE REGENERATION:
REVIEW ARTICLE, SV KHISTE ET AL
REVIEW OF CURRENT CONCEPT REVASCULARIZATION
/REVITALIZATION TUGBA BEZGIN DENTAL TRAUMATOLOGY 2015.
TISSUE ENGINEERING IN ENDODONTICS SHEHAB EL-DIN M. SABERET
AL JOURNAL OF ORAL SCIENCE, 2009

CURRENT OVERVIEW ON CHALLENGES IN REGENERATIVE
ENDODONTICS:: RAMTA BASAL ET AL:: J CONSERV DENT 2015
COMPARISON BETWEEN PRP , PRGF AND PRF: LIGHTS AND
SHADOWS IN THREE SIMILAR BUT DIFFERENT PROTOCOL
EUROPEAN REVIEW FOR MEDICAL ANDPHARMACOLOGICAL
SCIENCE2015;19:927-930
COMPARATIVE RELEASE OF GROWTH FACTORS FROM PRP, PRF,
AND ADVANCED-PRF SPRINGER JAN 2016
STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH- A BOON TO
DENTISTRY INTERNATIONAL JOURNAL OF ORAL HEALTH AND MEDICAL
RESEARCH | ISSN 2395-7387 | JULY-AUGUST 2016 | VOL 3 | ISSUE 2

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