Analysis
of the signal transduction pathways of human Dental Pulp Stem Cells during
osteogenic differentiation.

INTRODUCTION

In
recent years, there has been an increasing focus on mesenchymal stem cells
(MSCs) and their potential use in regenerative cell therapy. Researchers have
been able to isolate MSCs from various tissues of
the body, firstly from the bone marrow and later on in adipose tissue 1,2 umbilical cord
blood 3 and deciduous
teeth4 ; some possessing
more stem cells than others.

The dental pulp is
considered an abundant source of mesenchymal stem cells. Dental pulp stem cells
(DPSCs) possess the self-renewal capability, multi-lineage differentiation
capacity and clonogenic efficiency, characteristic for MSCs 5 Therefore, apart
from their biological function in odontoblast production and regeneration of
tooth structures such as dentine, they can be potentially useful in producing
other tissue cell types; notably osteocytes, myocytes, chondrocytes, adipocytes
and neuron-like cells under appropriate conditions. (*) According to previous
Hungarian results and international literature we know it’s possible for these
stem cells to be used for future regenerative purposes.

The ability of MSCs to
undergo osteogenic differentiation in vitro has been well established 6, however the
specific genes which are upregulated during this process are still not
completely understood.

This research will focus
on the differentiation of DPSCs along the osteogenic pathway, potentially
identifying those genes which are strongly upregulated.

The production of in
vitro cultures of osteogenic cells is fundamental to advancing our knowledge and
understanding of the physiological and pathological functions of the stomatognathic
system. From a dental point of view, a successful breakthrough could prove
useful in hydroxyapatite regeneration for tooth damage, bone grafts for
implantations, treatment of bone related diseases and orthopaedic surgeries and
congenital disorders such as cleft lip/palate patients, to name a few.

Studies have shown that
stem cells of dental origin are promising tools for cardiac repair following
myocardial infarction, treatment of spinal cord injury, neurodegenerative
diseases (e.g. Alzheimer’s disease) 7 and liver
fibrosis.

The aim of this research was
to:

–       
Establish primary cell cultures from the dental
pulp of healthy human adult wisdom teeth

–       
Identify multipotent stem cells in these
cultures

–       
To investigate the potential of pulpal
stem cells to undergo osteogenic differentiation in vitro by placing cells in
osteogenic inductive media using optimized pharmacological protocols.

–       
Analysis by high throughput screening
Taqman Array for specific osteogenic marker genes

–       
Comparison of the results with control
sample and identify which genes are upregulated and downregulated

 

 

 

 

 

 

 

 

 

BACKGROUND

Dental pulp

 

The
dental pulp is specialised loose connective tissue of ectomesenchymal origin comprised
of a neurovascular bundle, located centrally in the pulp cavity of every tooth.8  The pulp cavity is divided into the pulp
chamber (coronal pulp) and root canal (root pulp) and is enclosed by a rigid
chamber of dentin, which protects the pulp from external insults such as
mechanical trauma, chemical irritation or microbial invasion8.

Anatomically
and functionally, the dentin and pulp are considered to be a single entity,
made up of histologically distinct elements and often known as the dentin-pulp
complex (endodontium). Both tissues are derived from the dental papilla of the
tooth germ during tooth development.

Figure
1 – cross
section of an adult human molar (Encyclopedia Britannica , n.d.)

Development
of the teeth begins in the 6th week of intrauterine life and is
commonly divided into 4 key stages: Initiation, Bud stage, Cap stage, Bell
stage. The earliest sign of development is the presence of a primary epithelial
band – A thickening of the
ectodermal epithelium beneath the oral ectoderm 9 – of which the vestibular
lamina and dental lamina develop at the position of the future jaws. The
vestibular lamina  gives rise to the lips
and cheeks and is also responsible for the formation of the oral vestibule,
whereas the dental lamina forms the teeth.

Oral epithelial cells
near the basement membrane proliferate and rapidly invade the underlying
mesenchyme to give rise to the dental lamina – the origin of the tooth bud. This
is the beginning of the bud stage. The mesenchyme becomes more condensed with the
cells arranging in a more closely packed configuration around the epithelial
bud. The cap stage follows; marked by a concavity of the epithelium that
partially envelops the underlying mesenchyme10. The structure is
then widely referred to as the enamel organ. Within the concavity of the enamel
organ lies a condensation of ectomesenchymal cells known as the dental papilla.
An array of ectomesenchymal cells
called the dental follicle or sac surrounds the enamel organ at the periphery and
limits the dental papilla. These 3 structures together form one entity named
the tooth germ.

As the enamel organ and
dental papilla develop further, the epithelial bud assumes a characteristic
bell shape – Bell stage – where many changes occur, notably
histodifferentiation and morphodifferentiation11 . The enamel
organ is composed of inner enamel epithelium, outer enamel epithelium, stellate
reticulum, stratum intermedium and the enamel knot whereas the dental papilla
contains odontoblasts. These cells will differentiate, giving rise to
pre-ameloblasts and pre-odontoblasts in order to undergo polarization, leading
to the secretion of ameloblasts and odontoblasts. These cells will then go on
to form enamel and dentin, respectively. When dentin forms around the dental
papilla, the tissue in the centre is regarded as the pulp. The dental follicle
forms the cementum and supporting tissues (periodontium).

Each person normally has
52 pulp organs in their lifetime; 32 permanent and 20 deciduous. Histologically,
the pulp can be divided into different zones.

 1. Odontoblast layer – a layer of odontoblasts;
highly specialized cells on the pulp periphery that produce dentin throughout
the life of the tooth, which eventually causes the pulp to recede in size over
time.

2.
Cell poor zone – also known as Weil’s zone, the subodontoblastic layer that is
relatively free of cells. Capillary plexuses and small nerve fibres occupy this
zone however, as well as bundles of reticular (Korff’s) fibres12 that pass from
the central pulp area, through the cell-poor zone, in between the odontoblasts,
eventually incorporating into the matrix of the dentin layer.

3.
Cell rich zone – contains an abundance of fibroblasts, the principal cell type of
the pulp that synthesize and secrete type III collagen as well as ground
substance which form the body and integrity of the pulp organ. Undifferentiated
multipotent mesenchymal cells are also localised here. It is suggested that they
possess a connection with odontoblasts so that in cases of odontoblast injury
or death, signals can be generated and transmitted through these connection to
the mesenchymal cells and provoke their differentiation into odontoblasts or
odontoblast-like cells13

 4. Central pulp zone – contains the main
support system for the peripheral pulp and includes the extracellular matrix,
large vessels and nerves, with branches that provide nutrition to the critical
outer pulp layers.

In
addition to the above-mentioned cells, there are other cell types present in
the normal pulp. Immune cells such as polymorphonuclear leukocytes (most
commonly neutrophils), lymphocytes, plasma cells and plasma cells are not
present in large numbers in the normal pulp but their concentrations increase
in the presence of injury or inflammation.  Macrophages and histiocytes (fixed or
wondering) are associated with pulpal immunosurveillance and are highly
phagocytic, removing bacteria, foreign bodies, dead cells and debris. 13

The
primary function of the dental pulp is the formation and nutrition of dentin.
In addition to this, it serves to protect the vitality of the pulp by
containing a highly vascularised blood supply and innervation achieved by the
plexus of Raschkow, containing numerous unmyelinated and myelinated nerve
fibres, located just beneath the cell rich zone. 13 The pulp also
galvanizes odontoblasts into creating new reparative dentin in the face of
irritants as a means of defence.

 

 

Stem cells

      
i.         
Stem cells – General overview

 

Stem cells (SCs) are unspecialised progenitor cells, with the ability to
give rise to one or more types of mature specialised cells and also display
extensive unlimited, self-renewal potential. They can be classified
by the extent to which they can differentiate into different cell types, this
is defined as potency. Totipotent SC is the cell type with the greatest
differentiation potential, able to produce extra embryonic tissue as well as
every other cell type in a living organism. Spores and zygotes are examples of
totipotent SCs.14 When totipotent
SCs begin to specialize they become embryonic SCs. Embryonic SCs are isolated
and cultivated from the inner cell mass of the blastocyst of a growing embryo,
formed approximately 5 days after fertilization. These SCs exhibit pluripotency,
having lost the ability to form extra embryonic tissue but still retaining the
ability to differentiate into every type of adult cell, of which there are
around 200 types. 15

Tissue specific
non-embryonic stem cells (also referred to as somatic or adult stem cells) are
multipotent. They are more restricted in that they only differentiate into
cells of a particular lineage; cells of the specific tissue/organ in which they
reside. For example, blood forming (haematopoietic) SCs in bone marrow can only
give rise to red blood cells, white blood cells and platelets but not neurons16. Scientists
primarily work with these two naturally present types of SCs of the human body.
 

Induced pluripotent stem
cells (iPSC) were termed such after Shinya Yamanaka discovered in 2006 that
adult mouse skin fibroblasts can be reprogrammed genetically to an ESC-like
condition by introducing four genetic factors (cMyc, Oct3/4, Klf4 and Sox2) which provoked expression of marker
genes and growth factors important for maintaining the properties of ESCs.17 This has paved
the way for a new era in the stem cell therapeutics.

Throughout life we
continue to rely on capability of the stem cells to replace damaged tissues
thereby facilitating their repair.

All in all, SCs are key
tools helping researchers increase their knowledge about normal development,
disease onset and progression, especially regarding cancers, useful for
developing and testing new drugs and therapies for safety and effectiveness.

 

    
ii.         
Stem cells of dental origin and their markers

 

Dental stem cells (DSC) are
part of the group of adult stem cells known as mesenchymal cells. The other
type being haematopoietic stem cells. Research has shown that the neural
crest-derived DSC reside in various niches of deciduous and permanent teeth,
including the dental pulp of human exfoliated deciduous teeth 18and that of third
molars 5, the periodontal
ligament19 , the apical
papilla of immature teeth 20 and dental
follicle.21(Figure 2)22

In accordance with the
minimum standards, proposed by the International Society of Cellular Therapy
(ISCT) in 2006 to define human multipotent MSCs, DSCs express specific surface
markers such as CD90, CD105, CD73, Stro-1, Major Histocompatibility Complex I and
lack the expression of CD34, CD45, CD14 and Major Histocompatibility Complex II.
 The ability to rapidly adhere to
tissue-culture treated plastic under standard conditions and differentiate to
osteoblasts, chondroblasts and adipocytes in vitro is also a criterion of MSC. The
ISCT also termed MSCs as mesenchymal stromal cells, no matter the tissue from
which they are found.

Currently, several
ongoing studies using MSCs for the purpose of regenerative therapy. Takeda et
al demonstrated that the pluripotency of dental stem cells may be related to
the age of the tooth or the age of the donor.23 In other words,
deciduous teeth, molars, and wisdom teeth of young adults all contain sources
of dental stem cells with higher proliferation rates and potency than teeth of
older adults.

 

Figure 2
– The dental and associated tissues from
which unique populations
of dental MSCs can be found. Categorized according to their tissue of
origin. 
 
 
 
 

An
advantage of using DSCs is that it allows for autologous use because you’re
using the patient’s own tissues and thus eliminates likelihood of immunologic
reactions. DSC are also a good match for the entire family, risk of communicable
diseases can be avoided, easy accessibility, and treatment for organ scarcity
which is an expected future necessity24

 

One of the obstacles to
the far-reaching utilization of stem cells on a routine basis has been the
absence of easy accessibility to them, and the revelation of a good source of
easily acquired postnatal stem cells in teeth gives one a conceivable answer
for this issue.

Therefore, dentists may additionally turn out to be a critical first link in the chain
of informing their patients and accumulating dental stem cells, to be used not only for the therapy
of dental pathoses, but additionally for medical disease.

 

Figure 3

  
iii.         
Dental pulp stem cells

 

Dental pulp stem cells
(DPSC) are adult stem cells present inside the dental pulp of human permanent (DPSC)
and human exfoliated deciduous teeth (SHED). They were the first identified and
isolated adult dental stem cells by Gronthos et al in 20005. DPSC remain in a
quiescent state within the pulp until they are required, displaying their proliferative
and pluripotent characteristics by transforming
into odontoblasts, osteoblasts, and chondrocytes to produce dentin, bone, and
cartilage tissues respectively for the repair process. They also express
markers similar to those of bone marrow MSCs such as CD73, CD90, CD105 which helps
to confirm their identity as MSCs25

DPSCs and SHED have a typical fibroblast shape5 and are thought to preserve their plasticity up
to 25 passages during the cell culturing process26, which is good considering cell lines in continuous
culture are expected to suffer from genetic instability as the passage number
increases.

DPSC have shown why they are a good stem cell
contender for dental and medical tissue engineering. For instance, they form
bone when combined with platelet-rich plasma or hydroxyapatite27 can differentiate
into functionally active dopaminergic neuron-like cells 28and stimulate
angiogenesis and vasculogenesis in murine model of hind limb ischemia29 . DPSCs will be the
stem cell choice used in this research.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Osteogenic signalling pathways

Osteogenesis is the
production of bone. Bone is formed by one of two processes; endochondral
ossification which forms bone from a cartilage skeleton and intramembranous
ossification which forms bone through direct transformation of mesenchymal
tissue.  Osteoblasts are the main cell
component of bone. They are the committed bone precursor cells; products of
MSCs. They function in groups of connected cells and initially synthesize dense
crosslinked collage (usually type 1) and specialized proteins to a lesser
degree. These proteins are also considered bone associated markers – alkaline
phosphatase, osteocalcin, osteopontin and bone morphogenic proteins (BMPs), to
name a few. They compose the organic matrix of bone. Later on, osteoblasts function
in bone remodelling and mineral
metabolism.30

Osteoblasts, derived from
osteoprogenitor cells are found in large amounts in the periosteum, endosteum
and bone marrow.

Runx2 (also named CBFA1)
is a key osteoblast (and chondroblast) specific transcription factor, encoded
by the Runx2 gene, essential for differentiation of osteoprogenitor cells into
osteoblastic cell lineage and ultimately the formation of bone. During
osteoblast differentiation, osteoprogenitor cells express this transcription
factor as well as other factors like Osterix (Osx), Msx1, Msx2, at different
stages of differentiation.

Besides osteoblasts, MSCs
can also give rise to adipocytes. Existing experimental evidence suggests that
the adipogenic lineage has an inverse relationship with the osteogenic lineage.
In other words, an upregulation of adipogenic differentiation is associated
with a subsequent downregulation of osteogenic differentiation, or vice versa.31 Runx2 is considered
the master regulator of osteogenesis and target gene of various signalling
pathways including but not limited to ?-catenin
dependent Wnt, transforming growth factor-beta 1 (TGF-?1), Hedgehog and NELL-1 signalling, all of which follow
the inverse pattern mentioned above. 30

 

 

 

 

 

secreted
markers of osteoblasts

Osteocalcin

Osteocalcin (OC)
is a highly abundant non-collagenous protein found in bone and dentin. In
humans it is encoded by the bone
gamma-carboxyglutamic acid-containing protein (BGLAP)
gene.32 OC acts as a hormone and plays a role in bone
remodelling and mineralization, calcium ion homeostasis and energy metabolism.33  OC
constitutes 1-2% of the total bone protein 34. The gamma-carboxyglutmate residues are formed
by vitamin K dependent carboxylation and ensure the strong binding of OC to
calcium and hydroxyapatite (mineral component of bone).32  Secreted
solely by osteoblasts, OC is often used as a marker to measure osteoblast
activity.

Osteopontin

Osteopontin (OP) is an
extracellular structural protein encoded by the secreted phosphoprotein 1 (SPP1)
gene and part of the family of small integrin-binding ligand, N-linked
glycoprotein (SIBLING) proteins35. It’s suffix
-pontin signifies that OP has a role as a linking protein, for instance in the
attachment of osteoclasts to the mineralized bone matrix35. OP is synthesized
by a large number of tissues within the body including T-cells36, brain37, smooth muscle
cells38 together with
osteoblasts. This protein is also a proinflammatory cytokine that upregulates
the expression of interferon-gamma and interleukin-12 reducing production of interleukin-10 and is important in the
pathway that leads to type I immunity.