STEM CELLS IN PERIODONTICS

STEM CELLS
“Pro Life Paves Path For Life”
Dr Prajakta V Phadke

1. NEED FOR STEM CELLS IN PERIODONTICS
2.HISTORICAL BAKGROUND BEHIND THE DISCOVERY
3. WHAT ARE THE STEM CELLS ???
4. TYPES OF STEM CELLS
5. SOURCE AND DERIVATION OF VARIOUS STEM
CELLS
CONTENTS

INDIVIDUAL STEM CELLS
6. SHED : STEM CELLS FROM EXFOLIATED DECIDUOUS
TEETH
7. DFSC: DENTAL FOLLICLE STEM CELLS
8.DPSC : DENTAL PULP STEM CELLS
9.PDSC: PERIODONTAL LIGAMENT STEM CELLS
10. APICAL PAPILLA STEM CELLS
11. EMBRYONIC STEM CELLS

12. STEM CELL MARKERS FOR IDENTIFICATION
13. APPLICATION OF DENTAL STEM CELLS
14. CHALLENGES ENCOUNTERED
15. CONCLUSION

INTRODUCTION
NEED FOR STEM CELLS

 Periodontitis is a common and widespread disease in the oral
and maxillofacial region that causes the destruction of the
tooth-supporting tissues including alveolar bone, the
periodontal ligament (PDL) and root cementum.
 If left untreated, periodontitis will result in progressive
periodontal attachment and bone loss that may eventually
lead to early tooth loss .
 As a consequence, periodontal disease is one of the most
important concerns for dentists, patients and the public
dental healthcare system.

 Following disease control interventions such as tooth
cleaning/ scaling, root planning and periodontal
debridement,
 several procedures have thus far been attempted to achieve
periodontal regeneration, including bone graft placement,
guided tissue/bone regeneration (GTR/GBR) and the use
of various growth factors and/ or host modulating agents
(e.g., Emdogain_ and parathyroid hormone) .
 These techniques have proven somewhat effective in
promoting the reconstruction of the appendicular
musculoskeletal system.

 However, periodontal regeneration is especially challenging,
as it requires predictable regeneration of three quite
diverse and unique tissues (e.g., cementum, PDL, and bone)
and a triphasic interface between these different tissues to
guarantee the restoration of their complex structures.
 Unfortunately, current regenerative procedures that are used
either alone or in combination have limited success in
achieving this ambitious purpose, especially in advanced
periodontal defects

 Recent insights into the reparative capability of the
periodontium in conjunction with advances in stem cell
biology and regenerative medicine enable the development
of novel therapies using either endogenous regenerative
technology or cell-based therapeutics that are likely to
achieve robust regeneration with greater efficacy and
predictability .
 The acceleration of a patient’s endogenous regenerative
mechanisms that recruit host stem/ progenitor cells, a
biological process known as cell homing, for periodontal
regeneration has been considered as a highly useful and
practical approach for clinical utility

1908:A Russian histologist, named Alexander Maksimov, is the first one to
propose the term "Stem Cell". Maksimov proposed the term during a
congress of the hematologic society in Berlin. He was the first to
hypothesise the existence of haematopoietic stem cells.
1924: Alexander A. Maximow identifies a singular type of precursor cell
within the mesenchyme that develops into different types of blood cells.
The cells discovered, were later revealed to be mesenchymal stem cells.
1960s: Two scientists, Joseph Altman and Gopal Das, present scientific data
that indicate adult neurogenesis in the brain, suggesting the existence of
neural stem cells. Their findings back then, contradicted the widely
accepted "no new neurons" dogma of Cajal. As a result their work and
findings were largely ignored by the scientific community.
1963: James Edgar Till, along with Ernest McCulloch, are the first to illustrate
the existence of self-renewing cells in mouse bone marrow. They had
actually discovered the existence of hematopoietic stem cells.
1968: A bone marrow transplant is successfully used (for the first time)
between two siblings for the treatment of Severe combined
immunodeficiency (SCID).

1978: Haematopoietic stem cells are discovered in human cord blood.
1981: Martin Evans along with Matthew Kaufman, manage to extract mice
embryonic stem cells from mice blastocysts. They also cultured and
cultivated them. During the same year, Gail R. Martin almost
simultaneously illustrated various techniques for extracting mouse
embryonic stem cells. She is attributed for coining the "embryonic stem
cell" term.
1989: Sally Temple, describes the existence of multipotent, self-renewing
progenitor and stem cells in the subventricular zone (SVZ) of the mouse
brain.
1992: Brent A. Reynolds and Samuel Weiss manage to isolate neural stem
cells from the adult striatal tissue, including the SVZ of adult mice brain
tissue.

2001: Researchers of Advanced Cell Technology become the first
ones to clone early staged human embryos (at the stage of 4 to 6
cells)
2003: Songtao Shi, discovers that the primary teeth of children can be
used as a new source for extracting adult stem cells
2004–2006: In 2004 Hwang Woo-Suk announced the creation of
several human embryonic stem cell lines from unfertilised human
oocytes. It was later shown that his work was fabricated and no
human embryonic stem cell lines were actually produced
2005: Researchers from UC Irvine's Reeve-Irvine Research Centre
manage to partially restore mobility in paralysed rats, with
induced spine damage, by using neural stem cells.
April 2006: Scientists at the University of Illinois at Chicago identify
cord blood-derived multipotent stem cells with pluripotent
capacities

August 2006: Shinya Yamanaka becomes the first to derive induced
pluripotent stem cells from mice.
October 2006: Scientists at Newcastle University in England become
the first to differentiate umbilical cord blood stem cells into liver
cells
January 2007: A research team led by Anthony Atala discovers a new
type of stem cell, amniotic fluid stem cells (AFS cells). These stem
cells are found to be pluripotent in nature.
October 2007: The nobel prize for Physiology or Medicine goes to
Mario Capecchi, Martin Evans, and Oliver Smithies for their
pioneering work on mouse embryonic stem cells.
November 2007: Shinya Yamanaka again comes first. This time, for
being the first one to create human induced pluripotent stem
cells. James Thomson and his team comes close second, for the
same achievement.

 January 2008: Advanced Cell Technology researcher Robert Lanza
announces the first production of human embryonic stem cells that
didn't require the destruction of an embryo.
 March 2008: The first stem cell related study of succesfully
regenerated human knee cartilage is published. The study involves
the use of autologous mesenchymal adult stem cells.
 October 2008: A team from Germany led by Sabine Conrad creates
human pluripotent stem cells from spermatogonial cells of adult
testis. In the same month scientists created induced pluripotent stem
cells from a single human hair .
 November 2008: Paolo Macchiarini transplants the first human
organ, fully grown from stem cells. It was a trachea which was
transplanted on a Colombian female who had her own
collapsed due to tuberculosis.

 11 October 2010: The first human clinical trial involving embryonic stem
cells commences. The trial was later cancelled, supposedly for financial
reasons. As of today no info regarding the few treated patients has been
released.
 During the trial paraplegic patients with spinal cord injuries were
supposed to be treated using human embryonic stem cells. Only a handful
received the treatment prior to the trial's cancellation.
 June 2011:A team of Israeli scientists led by Inbar Friedrich Ben-Nun
produce stem cells from an endangered species. Their work has the
potential to one day save many different species that are in danger of
extinction.
 December 2012: Advance Cell Technology announces human stem cell
clinical trial

Three basic categories of cells make-up the human body:
germ cells, somatic cells and stem cells.
 Somatic cells include the bulk of the cells that make-up the
human adult and each of these cells in its differentiated state
has its own copy, or copies, of the genome; the only
exception being cells without nuclei, i.e. red blood cells.
 Germ cells are cells that give rise to gametes, i.e. eggs and
sperm.
 Stem cell is a cell with the ability to divide indefinitely in
culture and with the potential to give rise to mature
specialized cell types.

When a stem cell divides,
the daughter cells
 can either enter a path leading to the formation of a
differentiated specialized cell or
 self-renew to remain a stem cell, thereby ensuring that a pool
of stem cells is constantly replenished in the adult organ.
This mode of cell division characteristic of stem cells is
asymmetric and is a necessary physiological mechanism for
the maintenance of the cellular composition of tissues and
organs in the body.

A ‘‘stem cell’’ refers to "a clonogenic, undifferentiated cell that
is capable of self-renewal and multi-lineage differentiation".
(Smith A. A glossary for stem-cell biology)
In other words, a stem cell is capable of propagating and
generating additional stem cells, while some of its progeny
can differentiate and commit to maturation along multiple
lineages giving rise to a range of specialized cell types.
Depending on intrinsic signals modulated by extrinsic factors
in the stem cell niche, these cells may either undergo
prolonged self-renewal or differentiation

These essential attributes of ‘stemness’ are proposed to include:
(i) active Janus kinase signal transducers and activators of transcription,
TGFb and Notch signalling;[ DNA transcription – signalling]
(ii) the capacity to sense growth factors and interaction with the extracellular
matrix via integrins;
(iii) engagement in the cell cycle, either arrested in G1 or cycling;
(iv) a high resistance to stress with upregulated DNA repair, protein folding,
ubiquitination and detoxifier systems;
(v) a remodeled chromatin, acted upon by DNA helicases, DNA methylases
and histone deacetylases; and
(vi) translation regulated by RNA helicases of the Vasa type
Ramalho-Santos M, Yoon S, Matsuzaki Yet al. ‘Stemness’: transcriptional
profiling of embryonic and adult stem cells. Science 2002; 298: 597–600.

The defining features of stem cells include:-
a capacity to self-renewal and to undergo extensive
proliferation,
and the potential to reproducibly differentiate into functional
cells indicative of several different lineages

STEM CELL
CATEGORY
DEFINITION EXAMPLE
TOTIPOTENT
The capacity to differentiate
Into all possible cell types
including extra embyonic tissues
Fertilized egg
PLEURIPOTENT
The ability to differentiate into
almost all cell type. Pleuipotent
cells lack the capacity to contribute
to extraembryonic tissue and
therefore cannot develop into fetal
or an adult animal
Embryonic stem
cells
MUTIPOTENT
The potential to give rise to cells
from multiple , but limited amount
of lineages
Mesenchymal
stem cells
OLIGOPOTENT
The capacity to differentiates into
few cell type
Myeloid stem
cells
UNIPOTENT
The ability to differentiate into only
one type of cell
Skin

Totipotency
 is the ability to form all cell types of the conceptus, including
the entire fetus and placenta.
 Such cells have unlimited capability; they can basically form
the whole organism.
 Early mammalian embryos are clusters of totipotent cells.

Pluripotency
is the
ability to form several cell types of all three germ layers
(ectoderm, mesoderm and endoderm) but not the whole
organism.
In theory, pluripotent stem cells have the ability to form all the
200 or so cell types in the body.
There are four classes of pluripotent stem cells.
These are embryonic stem cells, embryonic germ cells,
embryonic carcinoma cells and recently the discovery of a
fourth class of pluripotent stem cell, the multipotent adult
progenitor cell from bone marrow.

Multipotency
 is the ability of giving rise to a limited range of cells and
tissues appropriate to their location, e.g. blood stem cells give
rise to red blood cells, white blood cells and platelets, whereas
skin stem cells give rise to the various types of skin cells.
 Some recent reports suggest that adult stem cells, such as
haemopoietic stem cells, neuronal stem cells and
mesenchymal stem cells, could cross boundaries and
differentiate into cells of a different tissue.
 This phenomenon of unprecedented adult stem cell plasticity
has been termed ‘transdifferentiation’ and appears to defy
canonical embryological rules of strict lineage commitment
during embryonic development **

SOURCE AND DERIVATION OF STEM
CELLS

Mammalian stem cells are usually classified according to their
tissue of origin.
The ovary and testis contain oogonia and spermatogonia, which
have been referred to as the stem cells of the gonads.
In adult mammals, only the germ cells undergo meiosis to
produce male and female gametes, which fuse to form the
zygote that retains the ability to make a new organism thereby
ensuring the continuation of the germ line.
In fact, the zygote is at the top of the hierarchical stem cell tree
being the most primitive and producing the first two cells by
cleavage

This unique characteristic of germ cells is known as
‘developmental totipotency’.
Intriguingly, Oct 4—an embryonic transcription factor critical
for the maintenance of pluripotency—continues to be
expressed in the germ cells but is absent in other peripheral
tissues.

In mammals, the fertilized egg, zygote and the first 2, 4, 8, and
16 blastomeres resulting from cleavage of the early embryo
are examples of totipotent cells.
Proof that these cells are indeed totipotent arises from the
observation that identical twins are produced from splitting of
the early embryo.
However, the expression ‘totipotent stem cell’ is perhaps a
misnomer because the fertilized egg and the ensuing
blastomeres from early cleavage events cannot divide to make
more of them.

Although these cells have the potential to give rise to the entire
organism, they do not have the capability to self renew and, by
strict definition therefore, the totipotent cells of the early embryo should
not be called stem cells.

Embryonic stem (ES) cells, however, are derived from the
isolated inner cell masses (ICM) of mammalian
blastocysts.
The continuous in vitro subculture and expansion of an
isolated ICM on an embryonic fibroblast feeder layer
(human or murine) leads to the development of an
embryonic stem cell line.
The cells of the ICM are destined to differentiate into
tissues of the three primordial germ layers
(ectoderm, mesoderm and endoderm)
and finally form the complete soma of the adult
organism.

 Adult stem cells—also known as somatic stem cells—can be
found in diverse tissues and organs.
 The best-studied adult stem cell is the hematopoietic stem
cell (HSC).
 Adult stem cells have also been isolated from several other
organs such as the brain (neuronal stem cells), skin
(epidermal stem cells), eye (retinal stem cells) and gut
(intestinal crypt stem cells)
.

Mesenchymal stem cells (MSCs) are another well characterized
population of adult stem cells.
It is thought that they respond to local injury by dividing to
produce daughter cells that differentiate into multiple
mesodermal tissue types, including bone, cartilage, muscle,
marrow stroma, tendon, ligament, fat and a variety of other
connective tissues.
The ease of culture has greatly facilitated the characterization
of MSCs.
In addition, recent studies have shown that the MSCs can also
differentiate into neuron-like cells expressing markers typical
for mature neurons, suggesting that adult MSCs might be
capable of overcoming germ layer commitment

STEM CELLS OF DENTAL TISSUE
ORIGIN
SHED : STEM CELLS FROM EXFOLIATED DECIDUOUS TEETH
DFSC: DENTAL FOLLICLE STEM CELLS
DPSC : DENTAL PULP STEM CELLS
PDSC: PERIODONTAL LIGAMENT STEM CELLS
APICAL PAPILLA STEM CELLS
EMBRYONIC STEM CELLS

1. STEM CELLS FROM HUMAN DECIDUOUS EXFOLIATED TEETH
Stem cells from human exfoliated deciduous teeth were first described by Miura et al. in 2003 as a unique stem cell population that was completely identified.
1. STEM CELLS FROM HUMAN DECIDUOUS
EXFOLIATED TEETH
Stem cells from human exfoliated deciduous teeth were first
described by Miura et al. in 2003 as a unique stem cell
population that was completely different from stem cells
previously identified.

The obvious advantages of SHEDs are:
a) Higher proliferation rate compared with stem cells from
permanent teeth; because they are less mature than other stem
cells found in the body.
b) Easy to be expanded in-vitro.
c) High plasticity since they can differentiate into neurons,
adipocytes, osteoblasts and odontoblasts.
d) Readily accessible in young patients.
e) Especially suitable for young patients with mixed dentition.
f) The process does not require a patient to sacrifice a tooth to
source the stem cells.
g) There is little or no trauma.
h) stem cells from human exfoliated deciduous teeth are
amenable to cryopreservation, meaning that these cells could
be stored for long periods of time on liquid nitrogen

SHED: Stem cells from human exfoliated deciduous teeth
Masako Miura
Proceedings of National Academy of Science of United States of America
SHED are distinct from DPSCs with respect to their higher proliferation
rate, increased cell-population doublings, sphere-like cell-cluster formation,
osteoinductive capacity in vivo, and failure to reconstitute a dentin–pulp-like
complex.
SHED apparently represent a population of multipotent stem cells that are
perhaps more immature than previously examined postnatal stromal stem-cell
populations
SHED could not differentiate directly into osteoblasts but did induce new
bone formation by forming an osteoinductive template to recruit murine host
osteogenic cells.
These data imply that deciduous teeth may not only provide guidance for
the eruption of permanent teeth, as generally assumed, but may also be
involved in inducing bone formation during the eruption of permanent
teeth.

Zheng et al. implanted stem cells derived from miniature
pig deciduous teeth into critical-sized bone defects
created in the parasymphyseal region of the mandible.
Their study demonstrated that the implanted stem cells
differentiated directly into new bone, resulting in the
formation of markedly more new bone in the defect site .
Furthermore,
Yamada et al performed allogeneic transplantation of dog
stem cells, in conjunction with platelet rich plasma,
into bone defects of the mandible.
The implanted cells generated well-formed mature bone
that was neovascularized in the defect sites

2.STEM CELLS FROM DENTAL FOLLICLE
(DFSC)
Teeth have the specific feature of being the only organ that
penetrates from the host’s internal tissue, ie, the jawbone,
through the “oral integumentary layer” and into the oral
cavity.
During root development, cementogenesis begins during root
formation. During this stage, the inner and outer enamel
epithelia fuse to form the bilayered Hertwig’s epithelial root
sheath (HERS), which then induces differentiation of DFSCs
into cementoblasts or osteoblasts

The DF is a loose connective tissue sac derived from ectomesenchymal
tissues. It surrounds the developing tooth and plays different roles during the
life of a tooth
The DF is formed at the cap stage of tooth germ development by an
ectomesenchymal progenitor cell population originating from cranial neural crest
cells .
 In addition to its function in periodontium development, the DF is also critical
for the coordination of tooth eruption .
During the tooth eruptive process, it remains adjacent to the tooth crown of
unerupted or impacted teeth .
 The DF also regulates osteoclastogenesis and osteogenesis for eruption.
 Alternatively, under pathological conditions, the DF can proliferate into
stratified squamous epithelium to generate dental cysts .
Hence, it has several key functions in both the development of the
periodontium and resorption of bone during tooth development.

 Park JY et al conducted a study to compare the
regenerative capacity of periodontal ligament stem
cells, dental pulp stem cells and periapical follicular
stem cells to see which cell population is the most
appropriate for clinical applications.
 The study was conducted on beagle dogs for a period of
8 weeks on apical involvement defect .
 The autologous periapical follicular stem cells
generated new cementum, alveolar bone and Sharpe's
fibers of periodontal ligament.
 However, periodontal ligament stem cells were found to
have more regenerative capacity.

 Guo et al. used dental follicle stem cells to assess the ability
of such cells to contribute to the formation of the tooth
root.
 They implanted dental follicle stem cells into three
different microenvironments in rats: the non mineralized
omental pocket, the highly mineralized skull and the
inductive alveolar fossa
 The dental follicle stem cells were implanted into these sites
in conjunction with a dentin matrix-treated scaffold. The
dental follicle stem cells contributed to dentin regeneration
within the omental pockets and contributed to mineralized
matrix formation in the skull defects.

 Interestingly, the dental follicle stem cells implanted in the
alveolar fossa contributed to the formation of root-like tissues
with a pulp–dentin complex and a periodontal ligament
connecting a cementum-like layer to host alveolar bone .
 These results demonstrate that the micro-environment into
which stem cells are implanted affects the capacity of these
cells to form differing tissues. More interestingly, these
results also demonstrate the potential that dental follicle
stem cells have in regeneration of tooth roots.

Schematic diagram of procedure used to
generate engineered dental root analogue.
The third molar tooth
is harvested from the mandible of a 6-month-old
pig to obtain dental follicle, dental pulp,
and enamel organ, and each cell is
independently isolated.
DFSCs are subcultured only until sufficient
cell numbers for periodontal tissue regeneration
are obtained.
The cylindrical bone cavity is made from pig
mandibular bone shaft.
Firstly, subcultured DFSCs are seeded at
the bottom of the bone cavity. Then, dental
pulp cells, enamel organ epithelial cells, and
subcultured DFSCs are successively placed
over the preceding layer.
A mimic of the tooth germ is thus created with
dental cell populations.
Dental follicle stem cells and tissue
engineering
Masaki J. Honda
Journal of Oral Science, Vol. 52, No. 4, 541-
552, 2010

3. ADULT DENTAL PULP STEM CELLS
(DPSC)
 Dental pulp is a highly vascularized tissue and contains
several niches of stem cells .
 The DPSC have multipotency, being capable of differentiating
into odontoblasts, osteoblasts, adipocytes, chondrocytes, or
neural cells.
 The regenerative capacity of the human dentin/pulp complex
implies that dental pulp may contain the progenitors that are
responsible for dentin repair.

Advantage Of Dental Pulp Stem Cells :
1. DPSC could regenerate a dentin-pulp-like complex, which is
composed of mineralized matrix with tubules lined with
odontoblasts, and fibrous tissue containing blood vessels in an
arrangement similar to the dentin-pulp complex found in
normal human.
2. DPSC posses striking features of self-renewal capability and
multilineage differentiation by finding that DPSC were capable
of forming ectopic dentin and associated pulp tissue in-vivo and
differentiating into adipocytes and neural-like cells.
Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo
S. Gronthos
PNAS:December 5, 2000 vol. 97

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal 4. PERIODONTAL LIGAMENT DERIVED STEM CELLS
The PDL is a specialized connective tissue, derived from dental
follicle and originated from neural crest cells.
The main features of the periodontal ligament are :-
rapid matrix turnover and
the ability to adapt to alterations in mechanical loading,
which, in combination
with the presence of heterogeneous cell populations, allows for
dynamic and strong connections between tooth root and bone, in
spite of the considerable force levels associated with mastication .
The ability of periodontal ligament to remodel and allow for tooth
movement is particularly important in the maintenance of the
periodontium.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Advantages of using periodontal ligament derived stem cells:-
1. MSC obtained from PDL - PDLSC are multipotent cells with
similar features of the BMMSC and DPSC, capable of
developing different types of tissues such as bone and tooth
associated tissues.
It was reported that PDLSC could differentiate into cells that
can colonize and grow on biocompatible scaffold, suggesting
an easy and efficient autologous source of stem cells for bone
tissue engineering in regenerative dentistry.

 2.Orciani et al verified the osteogenic ability of PDLSC and
pointed out that differentiating cells were also characterized
by an increase of Ca and nitric oxide production.
The authors demonstrated that local re-implantation of expanded
cells in conjugation with a nitric oxide donor could represent a
promising method for treatment of periodontal defects.
 3. Human PDL reveals itself as a viable alternative source
for possible primitive precursors to be used in stem cell
therapies.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Study was conducted by Sco B M et al .
Periodontal defect were surgically created on the buccal
cortex of the mandibular molar of immunodefecient rats.
Carrier used was Hydroxyapatite ⁄ β-tricalcium phosphate
particles.
After 6-8 weeks implanted periodontal ligament stem cells
demonstrated the ability to form cementum ⁄ periodontal
ligament-like structures and aid periodontal tissue repair.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Study was conducted by Liu X et al.
The defects in Periodontal lesion of the maxilla and mandibular first
molars of miniature pigs were treated with green fluorescent protein-labeled
periodontal ligament cells carrier being Hydroxyapatite ⁄ β-
tricalcium phosphate particles.
Transplanted green fluorescent protein-labeled periodontal ligament stem
cells had excellent capacity to form bone, cementum and periodontal
ligament when transplanted into a surgically created periodontal defect.
Green fluorescent protein-labeled cells were identified in the newly formed
bone, suggesting that the transplanted cells contributed to new-bone
formation.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Study was conducted by Kim S H et al .
to detect difference in regenerative potential between bone
marrow-derived mesenchymal stem cells and periodontal
ligament stem cells.
Saddle-like through-and through defects were treated with
PDLSC carrier being Hydroxyapatite ⁄ β-tricalcium
phosphate particles.
Transplantation of bone marrow-derived mesenchymal stem
cells and periodontal ligament stem cells into peri-implant
defects resulted in enhanced bone regeneration.
There was no significant difference in regenerative potential
between bone marrow-derived mesenchymal stem cells and
periodontal ligament stem cells.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal 6.EMBRYONIC STEM CELLS
In 1998, Thomson and co-workers derived the first human
embryonic stem (ES) cell line from the inner cell mass of 4- to
7-day-old blastocyst-stage embryos donated by couples
undergoing fertility treatment.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Defining properties of embryonic stem cells:-
1. Derived from the inner cell mass ⁄ epiblast of the blastocyst
of pre-implantation or peri-implantation embryo.
2. Capable of undergoing unlimited proliferation in an
undifferentiated state.
3. Exhibit and maintain a stable, diploid normal complement
of chromosomes.
4. Can give rise to differentiated cell types that are derivatives
of all three embryonic germ layers (ectoderm, mesoderm and
endoderm) even after prolonged culture.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal 5. Capable of integrating into all foetal tissues during
development.
6. Capable of colonizing the germ line and giving rise to egg or
sperm cells.
7. Clonogenic, i.e. a single ES cell can give rise to a colony of
genetically identical cells or clones, which have the same
properties as the original cell.
8. Expresses the transcription factor Oct-4, which then activates
or inhibits a host of target genes and maintains ES cells in
a proliferative, non-differentiating state.
9. Can be induced to continue proliferating or to
differentiate.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal 10. Lacks the G1 checkpoint in the cell cycle. ES cells spend
most of their time in the S phase of the cell cycle, during
which they synthesize DNA.
Unlike differentiated somatic cells, ES cells do not require
any external stimulus to initiate DNA replication.
11. Do not show X inactivation. In every somatic cell of a female
mammal, one of the two X chromosomes becomes
permanently inactivated but this does not occur in
undifferentiated ES cells.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal HURDLES ENCOUNTERED
 the clinical application of these unique cell types is currently
limited by two challenges: the difficulty of generating fully
functional cell types and safety concerns, particularly teratoma
formation.
 Another major obstacle is that human ESCs are isolated from
embryos, a procedure that ultimately leads to the destruction
of embryos and raises serious ethical concerns regarding the
moral status of the embryo, the sanctity of life and the
possible use of saviour siblings as a source of ESCs .

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal DENTAL STEM CELL MARKERS
Stem cell markers are genes and their protein products used by scientists
to isolate and identify stem cells. Stem cells can also be identified by
functional assays

DIFFERENTIATION
AND
TRANS DIFFERENTIATION

Differentiation is the process :-
whereby an unspecialized early embryonic cell acquires
the features of a specialized cell such as a heart, liver or muscle.
Differentiation in vitro can be spontaneous or controlled.
From a teleological perspective there appears to be no limit to
the types of cell that can be formed from hESC differentiation.
This is in contrast to the practical and theoretical constraints
levied on somatic stem cells by virtue of their position in
embryonic development.

 The possibility that cell fusion events might be an alternative
explanation for some remarkable reports of somatic stem cell
transdifferentiation has been highlighted by some studies.
Ying et al found that neural stem cells co-cultured with ES
cells could contribute to non-neural tissues not by
dedifferentiation but via fusion with the ES cells,
and Terada et al carried out similar co-culture experiments with
bone marrow cells and ES cells and found that the resulting
ES-like cells, which could differentiate to many different cell
types in vitro, were aneuploid

 The transdifferentiation phenomenon is not as straightforward
as it seems.
 currently there is no understanding of the developmental
mechanisms regulating transdifferentiation and its
physiological significance

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal APPLICATIONS OF DENTAL STEM
CELLS

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Gene and cell-based therapy
 The inherent proliferative and pluripotent capabilities of stem
cells may offer lifelong opportunities for treatment of some
important human diseases, including periodontitis, by repairing,
replacing or regenerating damaged tissues.
 Stem cells may act as suitable vehicles for the delivery of
therapeutic genes in gene therapy, and as therapeutic agents per
se in cell-based therapy.
 Gene therapy is a new approach for the treatment of human
diseases.
 It relies on genetic engineering, which involves molecular
techniques to introduce, suppress or manipulate specific genes,
thereby directing an individual s own cells to produce a therapeutic
agent.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  Two major strategies for delivering therapeutic transgenes
into human recipients are:-
(1) direct infusion of the gene of interest using viral or non-viral
vectors in vivo; and
(2) introduction of gene into delivery cells (often a stem cell)
outside the body ex vivo followed by transfer of the delivery
cells back into the body.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  The use of both in vivo and ex vivo gene delivery strategies
via adenoviral (Ad) vectors encoding growth promoting
molecules such as platelet-derived growth factor (PDGF) and
bone morphogenetic protein-7 has been investigated for its
potential in periodontal regeneration .
(Giannobile et al )
 Recent findings in rats have revealed sustained transgene
expression for up to 10 days at Ad-BMP-7 treated sites, and
enhanced bone and cementum regeneration at Ad-BMP-7 and
Ad-PDGF treated sites beyond that of control vectors.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  The introduction of transgenes into dental stem cells may offer an
alternative to conventional methods because stem cells have the
potential to provide a sustained source of growth factors for
regeneration.
 However, much work is still needed to optimize the number of
cells that are virally transuded to express specific genes, in order to
maximize the duration and extent of gene expression, and
ultimately to determine the success of gene transfer techniques in
periodontal regeneration.
 Further research is also needed to address potential risks of viral
recombination and immune responses towards viral antigens which
could potentially hinder the progress of gene therapy in treating
periodontal diseases.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  Banking teeth and dental stem cells offers patients a viable
alternative to using more invasive or ethically problematic
sources of stem cells, and harvesting can be done during
routine procedures in adults and from the deciduous teeth of
children.
 Now, dental professionals have the opportunity to make their
patients aware of these new sources of stem cells that can be
conveniently recovered and remotely stored for future use as
new therapies are developed for a range of diseases and
injuries.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal Craniofacial
regeneration
APPLICATION
OF
DENTAL STEM
CELLS
Cleft lip and
palate
Tooth
regeneration
Pulp
regeneration
Periodontal
ligament
regeneration
Enamel and
dentin
production

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal CHALLENGES ENCOUNTERED AND
FUTURE DIRECTIONS FOR
RESEARCH

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  BIOLOGICAL CHALLENGES
 Despite biological evidence showing that regeneration can
occur in humans, complete and predictable regeneration still
remains an elusive clinical goal, especially in advanced
periodontal defects.
 Periodontal regeneration, based on replicating the key cellular
events that parallel periodontal development, has not been
possible because of our incomplete understanding of the
specific cell types, inductive factors and cellular processes
involved in formation of the periodontium.
 Furthermore, most basic discoveries on periodontal stem cells
have emerged from cell culture and animal models which does
not always translate to the human situation.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  Thus, not all findings in animal models can be directly
extrapolated to humans. In addition, the molecular pathways
that underlie stem cell self-renewal and differentiation are
also largely unknown.
 Further research is needed to elucidate the cellular and
molecular events involved in restoring lost periodontal
tissues before a reliable biologically-based therapy can be
developed.
 In light of these concerns, the isolation and
characterization of stem cells from periodontal tissues may
provide a good starting point to investigate the role of stem
cells in periodontal wound healing and their potential
applications in regenerative therapy, including tissue
engineering.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal TECHNICAL CHALLENGES
 Biologically, the matrix scaffold should have good
biocompatibility for the cellular and molecular
components normally found in regenerating tissues.
 There is evidence to suggest that cultured human
PDLSCs in a suitable scaffold and implanted into
surgically-created periodontal defects can result in
the formation of a periodontal ligament-like
structure.
 However, the optimal mechanism of propagation
and incorporation of these cells into a carrier
scaffold still needs further refinement.

Park JY et al conducted a study to compare the regenerative capacity of periodontal ligament stem cells, dental pulp stem cells and periapical follicular The autologous periapical follicular stem cells generated new cementum, alveolar bone and Sharpe's fibers of periodontal ligament. However, periodontal  In addition, further studies are needed to understand the
conditions that induce lineage-specific differentiation and
efficacy of in vitro expanded stem cells derived from
regenerating periodontal defects.
 Possible karyotypic instability and gene mutations can
limit the usefulness of cell lines after prolonged culture.
 There are also difficulties in providing clinical-grade stem cell
lines using animal free media to prevent cross-infection in
humans.
 Thus, refinement of current techniques to facilitate
laboratory handling of these cells and to maximize their
regenerative potential represents a long-term endeavour if
these cells are to be used in clinical periodontics.

CLINICAL CHALLENGES
 There are a number of clinical barriers in MSC-based clinical
therapy that must be understood and overcome:
 immune rejection, tumour growth and efficacy of cell
transplantation.
 Firstly, it is important to understand how the immune system
will respond to human stem cell derivatives upon
transplantation.
 Generally, the immunogenicity of a human cell depends on its
expression of class I and II major histocompatibility
(MHC) antigens, which allow the body to distinguish its own
cells from foreign cells.

 Human ES cells express a low level of class I MHC antigens,
but this expression is up-regulated with differentiation.
 The use of patient-specific (autologous) adult stem cells from
redundant third molar teeth should overcome potential
immune rejection.
 However, this approach may be redundant if recent reports
are considered which indicate that MSC can suppress the
immune system and thus allows the use of either
autologous or allogeneic MSC preparations.

 Secondly, the prevention of tumour formation following MSC
implantation is a major safety consideration as current studies
lack sufficient statistical power and long-term follow-up to
draw firm conclusions.
 It is likely that the more specific and extensive the therapeutic
application, the longer the stem cells may have to remain in
vitro to obtain sufficient numbers for therapeutic use.

 Thus during this extended period in culture there could be a
greater likelihood that genetic or epigenetic changes will
accumulate. If such changes are not accompanied by an
overt phenotypic transformation, they may go undetected
and harm the patient.
 Therefore, it is critical to have a thorough understanding of
the rate of genetic change and the type of selective pressures
that allows this change to dominate a culture.

 Thirdly, it is unclear whether human stem cell derivatives
can integrate into the recipient tissue and Delivery of
appropriate cells and molecules to the target site without
inducing ectopic tissue formation is of paramount
importance for the safety and effectiveness of tissue
engineering-based periodontal regeneration.
 It is hoped that, as knowledge on progenitor cells, growth
factors and delivery systems improves, it will eventually lead
to the development of regenerative therapy based on
sound scientific principles

 The aim of regenerative medicine is to stepwise re-create in-vitro
all the mechanisms and processes that nature uses during
initiation and morphogenesis of a given organ.
 In this context, stem cell research offers an amazing potential for
body homeostasis, repair, regeneration and pathology. Many
agencies around the world are now funding stem cell research, and
growing numbers of scientists are entering this field.
 The result should be a global collaboration focused on delivering
clinical outcomes of immense benefit to the world’s population.
 We are just at the beginning of a very long road of work and
discovery, but one thing is certain - the research on stem cells – the
precursors for life is vital and must go on. Hence to conclude: “Pro-life
paves the path for life.”

STEM CELLS IN PERIODONTICS

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