stem cells in periodontics

STEM CELLS “Pro Life Paves Path For Life”

Dr Prajakta V Phadke

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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

HISTORICAL BACKGROUND

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

http://www.stemcellsfreak.com/p/embryonic-stem-cells.html

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.

http://www.stemcellsfreak.com/p/induced-pluripotent-stem-cells-ipscs.html



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.

http://www.stemcellsfreak.com/p/adult-stem-cells.html

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

http://www.stemcellsfreak.com/2012/12/act-announces-human-stem-cell-clinical.html

WHAT ARE STEM CELLS

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.

TYPES OF STEM CELLS

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

TOTIPOTENTThe capacity to differentiateInto all possible cell types including extra embyonic tissues

Fertilized egg

PLEURIPOTENTThe 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

MUTIPOTENTThe potential to give rise to cells from multiple , but limited amount of lineages

Mesenchymal stem cells

OLIGOPOTENTThe capacity to differentiates into few cell type

Myeloid stem cells

UNIPOTENTThe 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 different from stem cells previously identified.

1. STEM CELLS FROM HUMAN DECIDUOUS EXFOLIATED TEETHStem 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 previouslyidentified.

1. STEM CELLS FROM HUMAN DECIDUOUS EXFOLIATED TEETHStem 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 previouslyidentified.

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 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.

SHED: Stem cells from human exfoliated deciduous teethMasako MiuraProceedings of National Academy of Science of United States of America

http://www.pnas.org/search?author1=Masako+Miura&sortspec=date&submit=Submit

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

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The DF is a loose connective tissue sac derived from ectomesenchymaltissues. 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 toothis 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 atthe 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 engineeringMasaki 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 invivoS. GronthosPNAS:December 5, 2000 vol. 97

http://www.pnas.org/search?author1=S.+Gronthos&sortspec=date&submit=Submit

http://www.pnas.org/search?author1=S.+Gronthos&sortspec=date&submit=Submit


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 .[26] 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.


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.






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.


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.





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.





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.





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.





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.




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.




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.




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 .




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

http://en.wikipedia.org/wiki/Stem_cell

DIFFERENTIATION AND

TRANS DIFFERENTIATION

Differentiation is the process :-

whereby an unspecialized early embryonic cell acquiresthe 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

APPLICATIONS OF DENTAL STEM CELLS




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.




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.




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.




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.




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.




APPLICATION

OF

DENTAL STEM CELLS

Craniofacial regeneration

Cleft lip and palate

Tooth regeneration

Pulp regeneration

Enamel and dentin production

Periodontal ligament

regeneration




CHALLENGES ENCOUNTERED AND FUTURE DIRECTIONS FOR

RESEARCH




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.




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.




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.




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

CONCLUSION

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.”

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