Regenerative Dentistry::

A Reason to Smile through Stem Cells

By : Meera Nair C.S.R.D. Bhopal

SourcesSources Embryonic stem cells Adult stem cells Umbilical cord Amniotic fluid

Stem cells are ….

Capable of dividing and renewing themselves for long

periods without differentiating

Not fully specialized

Can give rise to specialized cells

Stem CellStem Cell

Differentiate

A Stem Cell can specialize into a

particular type of somatic cell

Self-renew

A Stem Cell can reproduce itself by

cell division

Self-renew

Differentiate

Stem Cell DifferentiationStem Cell Differentiation

Isolation of Stem CellsIsolation of Stem Cells

www.pall.com/images/StemCellGraphic.jpg

Found in adult tissue

Can self-renew many times

These are multipotent – they can differentiate to become only the types of

cells in the tissue they come from.

• Hematopoietic stem cells – give rise to blood cells

• Mesenchymal stem cells – give rise to cells of connective

tissues and bones

• Umbilical cord stem cells – a rich source of hematopoietic stem

cells

• Stem cells found in Amniotic Fluid – might be more flexible

than adult stem cells

ADULT STEM CELLS

Derived from Embryos

This stage embryo is called Blastocyst

~5 days old, a hollow microscopic

ball of cells

Can Self Renew

These are pluripotent- they can differentiate to become almost every

cell in the body Advantages of using Stem Cells:

– Can be programmed along one Developmental pathway

– Not likely to be rejected immunologically (fewer surface markers)

– May perhaps grow into new nerve or muscle cells

Embryonic Stem Cells

Stem cells derived from the inner cell mass of blastocyst stage human embryos have been shown to differentiate into several different cell types and have the potentials to one day replace or regenerate tissues

Krebsbach P.H., Robey P.G. (2002) Dental and Skeletal Stem Cells : Potential Cellular Therapeutics for Craniofacial Regeneration. Journal of Dental Education. Volume 66, No. 6 766-773

Two Sources of Embryonic Stem Cells

1. Excess fertilized eggs from IVF (In-vitro Fertilization) 1. Excess fertilized eggs from IVF (In-vitro Fertilization)

clinicsclinics

2. Therapeutic cloning (Somatic Cell Nuclear Transfer)2. Therapeutic cloning (Somatic Cell Nuclear Transfer)

Tens of thousands of frozen

embryos are routinely destroyed

when couples finish their

treatment.

These surplus embryos can be

used to produce stem cells.

Regenerative medical research

aims to develop these cells into

new, healthy tissue to heal

severe illnesses.

Stem Cells from Stem Cells from In vitroIn vitro-Fertilization-Fertilization

The nucleus of a donated egg is

removed and replaced with the

nucleus of a mature, "somatic cell" (a

skin cell, for example).

No sperm is involved in this process,

and no embryo is created to be

implanted in a woman’s womb.

The resulting stem cells can potentially

develop into specialized cells that are

useful for treating severe illnesses.

Somatic Cell Nuclear Transfer

Human Teeth are made up of soft

pulp that is a nonmineralized,

vascularized conjunctive tissue

with nutritional, sensorial, immune

and dentinogenic functions, and of

three different mineralized tissues:

dentin, cementum and enamel.

HUMAN TEETH

www.studiodentaire.com/en/glossary/pulp.php

Pulpal Stem Cells (PSCs)

Stem Cells from Human Exfoliated Deciduous teeth

(SHED)

Periodontal Ligament Stem Cells (PDLSCs)

Sources of Stem Cells from TeethSources of Stem Cells from Teeth

Dr. Songtao Shi, a Dentist and Researcher

working at the National Institute of Dental and

Craniofacial Research at the NIH, first

published that adult teeth contained stem cells.

He and others then went on to discover and

publish that children’s primary teeth also

contained stem cells, and that those cells

contained special properties. He named these

cells - Stem cells from Human Exfoliated

Deciduous teeth (SHED).

Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. 2003 SHED: Stem Cells from Human Exfoliated Deciduous Teeth. Proc Natl Acad Sci U S A. May 13;100(10):5807-12.

Dr. Songtao Shi

Pioneer Researcher………..Pioneer Researcher………..

Dental Pulp Stem Cells, or (DPSCs)

are multipotent stem cells that have

the potential to differentiate into a

variety of cell types (Gronthos et al.,

2000). Dental pulp is the part in the center

of a tooth made up of living soft

tissue and cells called odontoblasts.

Dental Pulp Stem Cells

DPSCs

Tissue similar to dentinOdontoblast-like cells PulpDentinEnamel

Cementum

PRODUCESPRODUCES

(Gronthos et al., 2002; Miura et al., 2003; Stevens et al., 2008).

DPSCs

Wide variety of other cell and tissue typesNeural cellsAdipocytesOsteoblastsChondrocytes Striated muscle

Differentiate

FeaturesFeatures

Ability to regenerate a Dentin-pulp-like complex in

an arrangement similar to the dentin-pulp complex

found in normal human teeth

Contain multipotent neural crest stem cells (NCSC)

Gronthos et al., 2000

Dentin-pulp-like complex is composed of :

Mineralized matrix with tubules lined with odontoblasts &

Fibrous tissue containing blood vessels.

Cytoarchitecture of a Dental Pulp Stem Cell

Cells selected for c-kit1, CD341 and STRO-11 were observed under a confocal

microscopy. The green fluorescence stains the cell cytoskeleton (revealed by

phalloidin); DAPI stains the nucleus.

The PDL is a specialized tissue located between the cementum

and the alveolar bone and has as a role the maintenance and

support of the teeth.

PDL contains STRO-1 positive cells that maintain certain plasticity

since they can adopt adipogenic, osteogenic and chondrogenic

phenotypes in vitro (Gay et al., 2007)

Periodontal ligament (PDL)

PDLSCs

Implanted into nude mice

Generated cementum/ PDL-like structures that resemble the native PDL as a thin layer

of cementum.

Four-week

Three-week

PDLSCs differentiated into Oil red-O-positive, lipid-laden adipocytes

PDLSCs have the potential for forming periodontal structures, including the cementum

and PDL.

Adipogenic-inductive cocktail

Osteo/odontogenic inductions

(Shi et al., 2002; Seo et al., 2004)

Alizarin-red-positive nodules were formed similar to MSCs & DPSCs

Exfoliated deciduous tooth houses living pulp remnants

consisting of connective tissue, blood vessels, and

odontoblasts.

12 to 20 cells from each exfoliated incisor formed adherent

colony clusters with extensive proliferative capacity (Miura et

al., 2003).

Ex vivo expanded SHED expressed STRO-1 and CD146

(MUC18), two early cell-surface markers for bone-marrow-

derived MSCs (Shi and Gronthos, 2003).

Stem Cells from Human Exfoliated Deciduous

Teeth (SHED)

Immuno-compromised mice

Hydroxyapatite/Tricalcium phosphate Hydroxyapatite/Tricalcium phosphate (HA/TCP) as a carrier(HA/TCP) as a carrier

SHED

Dentin-like structures are formed

Implanted

The Exfoliated Primary incisor contained dental pulp as shown (black triangles). The dashed line shows the occlusion edge of the incisor.

(B and C) Hematoxylineosin staining indicated dentin (De) Pulp of exfoliated deciduous teeth. (D) Single colonies were formed after SHED were plated at low density and cultured for 2 weeks. (E) SHED were capable of forming sphere-like clusters when cultured(F) The sphere-like clusters could be dissociated by passage through needles and subsequently

grew on 0.1% gelatin-coated dishes. (G) Proliferation rates of SHED, BMSSCs and DPSCs were assessed by BrdUrd (BrdU) incorporation for 12 h.

ee

e

Isolation of Isolation of SHEDSHED

Masako Miura, Stan Gronthos, Mingrui Zhao, Bai Lu, Larry W. Fisher, Pamela Gehron Robey, and Songtao Shi 2003. SHED:

Stem cells from human exfoliated deciduous teeth. Vol. 100 no. 10 5807–5812

Ex vivo expanded SHED expressed STRO-1 and CD 146 (MUC 18), SHED expresses a variety of osteoblast/odontoblastic markers, including alkaline phosphatase (ALP), matrix extracellular phosphoglycoprotein (MEPE), Bone sialoprotein (BSP), and DSPP.

STRO-1 is a cell surface protein expressed by bone marrow stromal cells and erythroid precursors.

MUC18/CD146, a pericyte marker

CONSTRUCTION OF A BIOENGINEERED TOOTH.

Single cell suspensions obtained from rat, pig or mice tooth germs

Seeded onto the surface of selected biomaterials (e.g. Collagen-coated

polyglycolic acid, calcium phosphate material, collagen sponges)

Re-implanted into the omentum of immunocompromised animals

Tooth is regenerated

Bioengineering of Tooth

Whole tooth regeneration by in vitro cell Manipulation has been carried out using

tooth germ cells.

Research on whole tooth regeneration is also advancing using a strategy of

transplanting artificial tooth germ and allowing it to develop in the adult oral

environment.

The cultured molar bud cells increased in number and were also able to form

bioengineered teeth

Kazuhisa Nakao, Takashi Tsuji. 2008. Dental regenerative therapy: Stem cell transplantation and bioengineered tooth replacement. Japanese Dental Science Review 44, 70—75

Regeneration of a whole tooth from bioengineered tooth germ in vitro and in vivo. (a) Phase contrast and histological images of the bioengineered tooth germ before and after 14 days of transplantation in a subrenal capsule. am: ameloblasts, BO: alveolar bone, bv: blood vessels, PD: pre-dentin, DE: dentin, E: epithelial cells, EN: enamel, M: mesenchymal cells, od: odontoblasts, p: pulp cells and PDL: periodontal ligaments. Scale bar: 100 mm. (b) Time course images of a bioengineered incisor (upper panel) and molar (lower panel) tooth germin an in vitro organ culture. Scale bar: 500 mm. (c) Separated primordia from bioengineered tooth germ that had been cultured for 2 days (left), and bioengineered tooth generated after 14 days of transplantation in extracted tooth cavity (right).

Potential cell source for Dental Regenerative Therapy

The healthy pulps of deciduous teeth are a

rich source of viable stem cells.

Pulp of deciduous teeth are highly

proliferative.

The ideal deciduous tooth for stem cell

recovery is a canine or incisor.

Supernumerary or mesodens are another

ideal source for dental stem cells.

Harvest Zone:

The harvest zone for stem cells is from the

deciduous canine to canine.

Deciduous Teeth

Whole or sectioned portions of third

molars contain healthy pulp and can be

recovered at the time of their removal.

Developing third molars have a larger

volume of pulpal tissue than teeth that

are mature with their roots completely

formed.

Wisdom TeethWisdom Teeth

It is best to recover these teeth during the developmental stage

(between 16-20 years of age), when the stem cells are very active

in the formation of the root and supporting root structures.

Third molars with healthy pulp can also be recovered later in life

and are always considered a source for viable stem cells.

All Permanent Teeth with healthy pulp are

potential sources of stem cells.

Stem Cells from within the pulp become

less proliferative as individuals

It is best to recover stems cells at the

earliest opportunity.

Permanent TeethPermanent Teeth

www.dailymail.co.uk/news/article-462335

Removal of parodontal tissue

Removal of apical and coronal part of the tooth Removal of dental pulp

Photo: Rodolfo Gonzales

Stem cells recovered from dental pulp can differentiate into bone, cartilage, and adipose cells in vitro (outside the body)               

MACS® MicroBeads are superparamagnetic particles that bind to specific antigens on the cell surface and magnetically label these cells. The MicroBeads do not alter structure, function, or activity status of labeled cells.

MACS MicroBeads are nano-sized

particles which are not detectable via

scanning electron micrograph. This image

shows a CD8+ T cell isolated with CD8

MicroBeads.

Small (50 nm) superparamagnetic particles coupled to highly specific

antibodies

Non-toxic and biodegradable

CHARACTERISTICS

Magnetic labeling

Cells are magnetically

labeled with microBeads

in a short incubation step

Magnetic separation

Cells are separated on a MACS

Column placed in a MACS

Separator. The flow-through can

be collected as negative fraction

depleted of the labeled cells.

Elution of the labeled cells

The MACS Column is removed

from the magnetic field. The

magnetically retained cells are

flushed out as positively selected

cells

1st Magnetic LabelingNon-target cells are magnetically labeled with a biotinylated antibody cocktail and Anti-Biotin MicroBeads.

1st Magnetic SeparationUndesired cells are retained in a MACS® Column placed in a MACS Separator while the unlabeled cells pass through.

2nd Magnetic LabelingTarget cells are magnetically labeled with MicroBeads according to a subset marker.

2nd Magnetic SeparationTarget cells are retained in the column while unlabeled cellspass through. After the column is removed from the separator, the target cells are eluted as the enriched, positively selected cell fraction.

Sequential Sorting

1st Magnetic labelingCells of interest are magnetically labeled with MultiSortMicroBeads.

1st Magnetic separationTarget cells are magnetically isolated by positive selection.

Release of Magnetic particlesMultiSort MicroBeads are enzymatically released.

Inhibition of Release reaction

2nd Magnetic labelingCell subset of interest is labeled with MACS® MicroBeadsaccording to a second marker.

2nd Magnetic SeparationTarget cells are separated.

MultiSort Strategy

I. Fluorescence-activated cell sorting (FACS)

Researchers use a FACS instrument to sort out the rare stem cells from the millions of other cells. 

Cells in suspension are tagged with fluorescent markers specific for undifferentiated stem cells

Labeled cells are sent under pressure through a nozzle and passed through an electric field

Cells are sorted according to the charge

CryopreservationCryopreservation

NEWS NEWS

On On

Use of Stem Cells in Teeth Use of Stem Cells in Teeth

Regeneration………Regeneration………

The researchers extracted

the groups of cells that would

go on to form teeth (called

the “tooth germ”) from

Embryonic mice.

Tooth Germs containing the

cells for building a tooth,

were transplanted into the

jaw bones of mice.

These Germs grew in to fully

functional teeth which were

similar to normal teeth in

terms of hardness and

response to pain stimulation

Clinical Trials

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