Regenerative Dentistry::

A Reason to Smile through Stem Cells

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

Stem Cell
Stem cells are .

Capable of dividing and renewing themselves for long

periods without differentiating

Not fully specialized Can give rise to specialized cells

Sources Embryonic stem cells Adult stem cells Umbilical cord Amniotic fluid

Self-renew

Differentiate

Self-renew

Differentiate

A Stem Cell can reproduce itself by

cell division

A Stem Cell can specialize into a

particular type of somatic cell

Stem Cell Differentiation

Isolation of Stem Cells

www.pall.com/images/StemCellGraphic.jpg

ADULT STEM CELLS

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

Embryonic 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

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) clinics

2. Therapeutic cloning (Somatic Cell Nuclear Transfer)

Stem Cells from In vitro-Fertilization

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.

Somatic Cell Nuclear Transfer

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 womans womb.

The resulting stem cells can potentially develop into specialized cells that are useful for treating severe illnesses.

HUMAN TEETH

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.

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

Sources of Stem Cells from Teeth

Pulpal Stem Cells (PSCs) Stem Cells from Human Exfoliated Deciduous teeth

(SHED)
Periodontal Ligament Stem Cells (PDLSCs)

Pioneer Researcher..

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

Dr. Songtao Shi

publish that childrens 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.

Dental Pulp Stem Cells

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.

DPSCs
PRODUCES

Odontoblast-like cells

Pulp Dentin Enamel Cementum

Tissue similar to dentin

DPSCsDifferentiate

Wide variety of other cell and tissue types Neural cells Adipocytes Osteoblasts Chondrocytes Striated muscle

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

Features

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)

Dentin-pulp-like complex is composed of : Mineralized matrix with tubules lined with odontoblasts & Fibrous tissue containing blood vessels.
Gronthos et al., 2000

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.

Periodontal ligament (PDL)

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)

PDLSCs (Shi et al., 2002; Seo et al., 2004) Implanted into nude mice

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

Adipogenic-inductive cocktail

Three-week Four-week

PDLSCs differentiated into Oil red-O-positive, lipid-laden adipocytes Osteo/odontogenic inductions

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

PDLSCs have the potential for forming periodontal structures, including the cementum and PDL.

Stem Cells from Human Exfoliated Deciduous Teeth (SHED)

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-marrowderived MSCs (Shi and Gronthos, 2003).

SHED

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

Immuno-compromised mice

Dentin-like structures are formed

Isolation of SHED
e e

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.
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 58075812

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

MUC18/CD146, a pericyte marker

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.

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, 7075

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

Deciduous Teeth

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.

Wisdom 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. 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.

Permanent Teeth

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.

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

Photo: Rodolfo Gonzales

Removal of parodontal tissue

Removal of apical and coronal part of the tooth

Removal of dental pulp

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.

CHARACTERISTICS

Small (50 nm) superparamagnetic particles coupled to highly specific

antibodies Non-toxic and biodegradable

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

Elution of the labeled cells

The MACS Column is removed from the magnetic retained field. cells The are magnetically cells

be collected as negative fraction

depleted of the labeled cells.

flushed out as positively selected

Sequential Sorting
1st Magnetic Labeling Non-target cells are magnetically labeled with a biotinylated antibody cocktail and Anti-Biotin MicroBeads. 1st Magnetic Separation Undesired cells are retained in a MACS Column placed in a MACS Separator while the unlabeled cells pass through.

2nd Magnetic Labeling Target cells are magnetically labeled with MicroBeads according to a subset marker.
2nd Magnetic Separation Target cells are retained in the column while unlabeled cells pass through. After the column is removed from the separator, the target cells are eluted as the enriched, positively selected cell fraction.

MultiSort Strategy
1st Magnetic labeling Cells of interest are magnetically labeled with MultiSort MicroBeads. 1st Magnetic separation Target cells are magnetically isolated by positive selection. Release of Magnetic particles MultiSort MicroBeads are enzymatically released.

Inhibition of Release reaction

2nd Magnetic labeling Cell subset of interest is labeled with MACS MicroBeads according to a second marker.

2nd Magnetic Separation Target cells are separated.

I. Fluorescence-activated cell sorting (FACS)

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

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

Cryopreservation

NEWS On Use of Stem Cells in Teeth 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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