Tooth Tissue Engineering: Potential and Pitfalls

Abstract

All over the world a large number of people suffer from tooth diseases like dental caries, tooth abscess, and plaques. Tooth loss or damage, which occurs frequently in our society are generally repaired by applying several conventional methods, such as root-canal treatment, direct pulp capping and dental implants. These methods are quite painful, create damage to the surrounding tooth tissues and also may at times have adverse side-effects. The limitations of the conventional methods can be overcome by applying the concept of tooth tissue engineering. Tooth tissue engineering is the application of biosciences and engineering to regenerate a biofunctional tooth, which can be used to replace the missing tooth or repair the damaged tooth. Tissue engineering involves three key elements - cell, scaffold and growth factors, which interact with each other to regenerate a specific tissue. The success of tissue engineering depends on the proper selection of these three key elements and understanding the interactions among them. To bring us close to the realization of a tissue-engineered tooth, immense progress is going on in understanding how tooth is first developed, and there is a good advancement in tooth regeneration. In this review, “tooth tissue engineering” will be discussed, along with the recent advancements and challenges in bring a biofunctional tooth from laboratory out into clinical use.

Full text

Tooth Tissue Engineering: Potential and Pitfalls
Sweta Gupta1,a, Chhavi Sharma1,b, Amit K. Dinda2,c, Amiya K. Ray1,d,
Narayan C. Mishra1,e*
1 Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur Campus,
Saharanpur-247001, U.P, India
2 Department of Pathology, All India Institute of Medical Sciences, New Delhi - 110029, India
a swetag009@gmail.com, b chhavisharma19@gmail.com, c amit_dinda@yahoo.com,
d amiyakumarray@gmail.com, e mishrawise@googlemail.com,
Keywords: Tooth; scaffold; tissue engineering; biomaterials; growth factors; stem cell.
Abstract. All over the world a large number of people suffer from tooth diseases like dental caries,
tooth abscess, and plaques. Tooth loss or damage, which occurs frequently in our society are
generally repaired by applying several conventional methods, such as root-canal treatment, direct
pulp capping and dental implants. These methods are quite painful, create damage to the
surrounding tooth tissues and also may at times have adverse side-effects. The limitations of the
conventional methods can be overcome by applying the concept of tooth tissue engineering. Tooth
tissue engineering is the application of biosciences and engineering to regenerate a biofunctional
tooth, which can be used to replace the missing tooth or repair the damaged tooth. Tissue
engineering involves three key elements - cell, scaffold and growth factors, which interact with each
other to regenerate a specific tissue. The success of tissue engineering depends on the proper
selection of these three key elements and understanding the interactions among them. To bring us
close to the realization of a tissue-engineered tooth, immense progress is going on in understanding
how tooth is first developed, and there is a good advancement in tooth regeneration. In this review,
“tooth tissue engineering” will be discussed, along with the recent advancements and challenges in
bring a biofunctional tooth from laboratory out into clinical use.
Introduction
Tooth is a small calcified structure, found in the entrance of the alimentary canal of both
invertebrates and vertebrates. It aids in grasping and processing of food, providing defense from
any attack, giving shape to the face and aiding clear speech. Presence of teeth also helps to prevent
dementia as mastication stimulates the brain [1]. Tooth plays an important role in overall health
and activity. Tooth loss or damage is quite frequent. All over the world, a large amount of
population suffers from tooth diseases like dental caries (cavities, holes in the teeth), tooth abscess,
and plaques. 20% of all inherited disorders are accounted for by tooth congenital abnormalities. In
the western world, 85% of adults have had some dental treatment and 7% have lost one or more
teeth by the age of 17 [2]. Tooth loss is a problem of great magnitude since approximately 35% of
the world population is edentulous by the age of 65. World Oral Health Report 2003, identified
dental caries as a continuing global problem, with an estimated five billion people worldwide,
having experienced the disease. Treatment costs are estimated as accounting for some 5–10% of
the healthcare budgets in industrialized countries [3]. Tooth imposes an incredible amount of
importance in our life although it is not essentially required for our survival. Missing tooth
adversely affects mastication, articulation, facial esthetics and psychological health, and it can result
in difficulty in movement of the remaining teeth and a lack of self-confidence. Though these are
not life-threatening, but the quality of daily life is adversely affected. It leads to physical and
mental suffering that can markedly lower an individual's quality of life. Loss or damage of tooth is
mainly because of dental wear lesions (abrasion, attrition, and erosion), bacterial infections,

chemicals, trauma, fractures and genetic defects. The major acquired pathological conditions that
affect tooth, concern the erosion of non-living elements (enamel and dentin), damage to living
tissues (pulp and periodontium) and the loss of whole tooth.
To repair the loss or damage of tooth, several conventional methods are applied, which include
extrinsic dental interventions such as tooth transplantation (allotransplantation, autotransplantation),
root-canal treatment, direct pulp capping, artificial dentures and dental implants (metal like titanium
or ceramics) [4]. But these methods are quite painful, create damage to the surrounding tooth
tissues and also sometimes have adverse side-effects because of several reasons, like
incompatibility between dental tissues and non-biological substitutes, insufficiency of host bone in
the jaw to accommodate the implant, poor mechanical properties of the periodontium and lack of
immunological properties of the crevicular sulcus [5, 6]. In addition, these approaches fail quite
frequently and have considerable costs. Even if the outcome is good, pathological repeats are
common [5, 6]. Therefore, there is a need for a better, biologically-oriented therapeutic approach,
which is “tooth tissue engineering” or “regeneration of biofunctional tooth”. Tooth tissue
engineering is the application of our knowledge of biosciences and engineering to regenerate a
biofunctional tooth, which can be used to replace the missing tooth by a bio-functional one or repair
the damaged tooth.
In this review, a concise part of the tooth anatomy, morphology and its development is
discussed. It is then followed by discussing the tissue engineering approaches to repair tooth and
describes the three key elements of tooth tissue engineering, i.e. dental stem cell sources, scaffold
biomaterials and the growth factors required for tooth tissue regeneration. Also discussed is recent
endeavours in tooth tissue engineering as well as the prospective outcome of the current
advancement in this line of research. Finally, the article will highlight significant challenges and
hurdles in bringing tooth tissue engineering out from laboratory to clinical use.
Tooth – A Brief Overview of Basic Anatomy, Morphology and Tooth Development
Knowledge of tooth anatomy is necessary for understanding the pathological process and the
possible therapies. Due to different dietary habits and ecological adaptations, the tooth of
vertebrates acquires numerous anatomical forms and shapes such as incisors (cutting teeth), canines
(tearing teeth), premolars and molars (grinding teeth). These different shapes of teeth help in
protecting oral mucosa physiologically and aid to reduce stress forces on the tooth and the alveolar
bone. A whole tooth is divided into two parts - the crown and the root (Figure 1). The line of
junction between the crown and the root is called the cementum-enamel junction (cej - neck). The
crown emerges from the alveolar bone and is covered by enamel. The root is inserted into the bone
and is coated by cementum.
Although the morphology of tooth varies depending on species and location, but they are similar
in structure being organized into 3 parts - enamel organ (enamel), dental papilla (dentin and dental
pulp) and dental follicle (cementum, alveolar bone and periodontal ligament) (Figure 1). The hard
part of the tooth is composed of three different calcified tissues, enamel, dentin and cementum,
where enamel is the hardest of all these three. The dental pulp is the soft part and fills the centre of
the tooth. Tooth structural composition provides durability for a lifetime despite the enormous
abrasive forces, large temperature changes, varying pH and a moist and corrosive environment to
which teeth are continuously exposed [7]. The structural composition, physical properties and
different functions of tooth has been summarized in Table 1.
The morphological stages of tooth development consist of early bud stage, cap or proliferative
stage and bell stage, where tooth histo-differentiation and morpho-differentiation occurs, as
illustrated in Figure 2. Tooth development is characterized by highly orchestrated mutual inductive
interactions between two major cell types: stomodeal ectoderm (epithelial) cells and cranial, neural
crest-derived ectomesenchyme cells, which result in differentiation and spatial organization of cells
to form tooth-organs [13]. Stomodeal ectoderm cells form ameloblasts, which contributes to

prepare enamel tissues, whereas ectomesenchyme cells form odontoblasts, pulp cells,
cementoblasts, osteoblast and fibroblast (Table 2). Dental mesenchyme is termed
‘ectomesenchyme’ due to its earlier interaction with the neural crest [14]. These two types of cells,
stomodeal ectoderm cells and ectomesenchyme cells are always required for the growth of tooth
and the source of these cells is an important issue in tooth regeneration.
Figure 1. Anatomy of the tooth.
Figure 2. Schematic illustration of the main stages of tooth development: Oral epithelium
(ectoderm) forms a thickened epithelial band, dental lamina (a) budding of this epithelium towards
the underlying cranial neural crest-derived ectomesenchyme starts at the sites of future primary
teeth. Morphogenesis proceeds through the bud (b) and cap stages (c) to the bell stage (d) where
differentiation of odontoblasts (mesenchymal) and ameloblasts (epithelial) takes place. Finally
developing tooth starts erupting into the mouth (e).

Table 1. Structural composition, physical properties and functions of tooth tissues.
Tooth Tissue
Physical
Properties
Component
Protein
Cell constituents
Functions
Ref.
Enamel
Translucent,
vary in colour
from yellowish
to greyish
white, non-
living tissue
96%
inorganic
matter*,
1% organic
matter, and
3%water
Amelogenin
(90%),
ameloblastin,
enamelin and
tuftelin
Formed by dental
epithelial cells
(ameloblasts)
Endure crushing
pressure, produces a
cushioning effect of the
tooth's different
structures, and protects
the tooth from the wear
and tear of chewing.
[8]
Dentin
Light yellow,
calcified living
tissue, more
radiolucent
than enamel
and very
porous
70%
inorganic
matter*,
20% organic
matter, and
10%water
Dentin
sialoprotein,
dentin
phosphoprotein
and
dentin matrix
protein I
Formed by
odontoblasts that
line the walls of the
pulp cavity.
Living tissue transmits
pain stimuli by the way
of dentinal fibres.
[9]
Dental pulp
Hollow space
within the
dentin
Coronal pulp,
and Radicular
pulp
_
Composed of dental
mesenchymal cells
including putative
dental stem cells
(DSCs), fibroblasts,
macrophages and T
lymphocytes
Furnishes nourishment
to the dentin; provides
sensation to the tooth,
and responds to
irritation, either by
forming reparative
secondary dentin or by
becoming inflamed.
[10]
Cementum
Mineralized
tissue, light
yellow in
colour, covers
the roots of the
tooth in a thin
layer
45%
inorganic
matter*, 55%
organic
matter
Type I collagen
proteins
Formed by
precementoblast
Serves as an attachment
surface to anchor the
tooth to the bony walls
of the tooth sockets in
the periodontium and
protect the root.
[11]
Periodontium
Connective
tissues that
attach the
cementum to
the alveolar
bone via the
Sharpey's
fibres.
_
_
Osteoblasts and
osteoclasts present
on the alveolar
bone, fibroblasts,
epithelial cell rests
of Malassez, and
macrophages.
Support, protect and
provide nourishment to
the tooth, enable
sensory perception and
cushion mechanical
forces during
mastication as it has
shock absorbing
capacity.
[12]
* Inorganic matter - Calcium and Phosphorus as in hydroxyapatite
Table 2. Summary of derivation of tooth tissues.
Tooth part
Origin
Tooth Tissues formed
Cells involved
Enamel organ
Ectoderm
Enamel
Ameloblast
Dental papilla
Condensed
inner mass of
ectomesenchyme
Dentine
Dental pulp
Odontoblast (outer cells
of dental papilla)
Central cells of dental
papilla
Dental follicle, or
Dental sac
Remaining
ectomesenchyme
surrounding outside the
enamel
Cementum
Alveolar bone
Periodontal Ligament
Cementoblast
Osteoblast
Fibroblast

Tissue Engineering Approach to Repair Tooth
Langer and Vacanti defined tissue engineering as an “interdisciplinary field that applies the
principles of engineering and life sciences toward the development of biological substitutes that
restore, maintain, or improve tissue function” [15]. Tooth tissue engineering may be described as
the regeneration of tooth by the application of our knowledge of biosciences and engineering. For
successful tooth tissue engineering, three prerequisites are needed: (1) sufficient numbers of cells
with odontogenic capacity; (2) an appropriate scaffold, a 3D porous architecture, to seed the cells;
and (3) factors to stimulate odontogenic differentiation in vivo. Despite of tooth's structural
complexity, the advancement of biomedical engineering techniques has given rise to two currently
employed approaches for tooth regeneration (Figure 3). The first approach is to regenerate tooth by
seeding stem cells in scaffolding biomaterials. The cell-seeded scaffold guides and supports tooth
formation. This technique has shown promising results in regeneration of the periodontium. The
second approach attempts to reproduce or mimic the developmental or “organotype” approach that
facilitates the development of tooth from epithelial and mesenchymal (stem) cells, either from tooth
germs or other sources. This approach uses embryonic tissues (dental epithelium and dental
mesenchyme) harvested from a mouse foetus and direct the formation of organized tooth through
epithelial-mesenchymal interactions in organ culture. It requires an understanding of the principles
that regulate early tooth development in the embryo [16]. The first method is more convincing
since its key elements are more controllable.
With the emerging concepts of tissue engineering, biological tooth may become an alternative
for replacing missing tooth. For biological replacement of tooth tissues or whole tooth, both in vivo
and in vitro, new strategies have been designed [17]. A major move ahead would be to reconstruct
tooth in patients without adequate bone support [18]. To grow replacement tooth in the individual’s
own mouth, would be a highly attractive therapy to permanently replace tooth in situ. Dentistry has
taken the same approach to tooth decay-filling cavities for decades, but new techniques for
rebuilding tooth from inside out could transform the profession over the next decade [19]. An
ambitious dream of numerous dentists is to be able to substitute the artificial material with a
biological, cell-based one that is able to form a genuine replica of the damaged tooth part or the
entire lost tooth. It should lead to a permanent solution to the damaged tooth without the need for
supplementary therapies, thus making it a cost-effective treatment in the long term.
Stem Cells used in Tooth Tissue Engineering
Stem cells have a self-renewing capacity that can regenerate any tissue for life time, and because of
this key property the stem cells are being used in tissue regeneration. Tooth organ contain two
types of stem cells ― Embryonic Stem (ES) cells and Adult Stem cells. Pluripotent ES cells are
derived from the inner cell mass of mammalian blastocysts and can be maintained indefinitely in
culture. When injected back into blastocysts, these cells contribute to all tissues, including germ
cells [20]. Thus, ES cells are a renewable source of cells that can be stimulated to differentiate into
precursors of any cell type. Adult dental stem contains several populations of stem cells, which
have been differentiated into two categories on the basis of their origin, that is whether they are
derived from mesodermal layer (dental ectomesenchymal stem cells) or ectodermal layer (dental
epithelial stem cells) of the embryo (Box 1). The postnatal epithelial and ectomesenchymal dental
stem cells present in immature tooth buds have demonstrated the ability to generate bioengineered
and anatomically correct but miniature-sized tooth crowns containing enamel, dentin, pulp and
alveolar bone [21].
Adult dental stem cells can be isolated at various stages of the organogenesis. Stem cells from
apical papilla (SCAP) and dental follicle precursor cells (DFPC) are present in tooth where
developmental program continues, whereas periodontal ligament stem cells (PDLF), dental pulp
stem cells (DPSC), and stem cells from human exfoliated deciduous tooth (SHED) represent the
cells present in tooth (Figure 4) which have already completed their development [22]. Dental stem

cells can also be isolated according to their anatomical locations, colony-forming ability, expression
of stem cell markers, and regeneration of pulp/dentin structures in vivo. Tooth tissue engineering
using stem cells is based on their isolation, association and culture as recombinants in vitro or ex
vivo conditions to assess tooth morphogenesis and cell differentiation into tooth specific cells that
will form enamel, dentin, cementum and alveolar bone. Dental stem cells are multipotent stem cells
having the future potential to differentiate into a variety of other cell types (Table 3) including
cardio myocytes to repair damaged cardiac tissue following a heart attack [23], neuronal to generate
nerve and brain tissue [24], myocytes to repair muscle [25], osteocytes to generate bone [26],
chondrocytes to generate cartilage and adipocytes to generate fat.
Dental tissues are specialized tissues that do not undergo continuous remodelling as shown in
bony tissues. Therefore, stem/progenitor cells derived from these tissues may be more committed
or restricted in their differentiation potency in comparison with bone marrow mesenchymal stem
cells (BMMSCs). The use of dental stem cells has been increased significantly over the past 15
years. Stem cells of dental origin can be used for various functions in dentistry, e.g., intended for
development of new tooth, management of periodontal diseases, for cleft palate treatment,
dentofacial orthopaedics corrective methods and geno-dental interrelations [35, 36].
Figure 3. Two current ways of tooth tissue engineering (a) Dissociated tooth germs seeded onto a
tooth-shaped scaffold, produced small complex tooth-like structures; (b) Epithelial and
mesenchymal (stem) cells, either from tooth germs or other sources, grown in organ culture and
form organized tooth through epithelial-mesenchymal interactions.

Box 1. Dental stem cells for tooth tissue engineering.
Figure 4. Derivation of Dental derived Stem cells. (a) Dental Follicle Progenitor cells (DFPCs)
isolated at early stage before tooth eruption. (b) Dental Pulp Stem cells (DPSC), Periodontal
Ligament Stem cells (PDLSC), and Apical Papilla Stem cells (APSC) isolated from permanent
tooth.

Table 3. Different stem cells with their differing degrees of proliferative potential and the
differentiation potency [27, 21].
Cell Type
Proliferative
potential
Differentiation Potency
Ref.
In vitro Analysis
(Multipotentiality)
In vivo Analysis (Ectopic
tissue formation)
DPSCs
++++
Osteo/Dentinogenic, Adipogenic,
Chondrogenic, Myogenic,
Neurogenic
Dentin-pulp-like complex,
Odontoblast-like cells, bone-
like tissue
[28]
SHED
++++++++
Dentinogenic, Adipogenic,
Chondrogenic, Myogenic,
Neurogenic, Osteoconductive
Dentin-pulp-like complex,
Odontoblast-like cells, No
dentin-pulp complex
formation, Bone formation
[29]
SCAP
++++
Dentinogenic, Adipogenic,
Chondrogenic, Myogenic,
Neurogenic
Dentin-pulp-like complex,
Odontoblast-like cells
[30]
PDLSCs
ND
Osteo/Cementogenic, Adipogenic,
Chondrogenic, Myogenic,
Neurogenic
PDL like formation,
Cementum like formation
[31]
DFPCs
ND
Cementogenic, Odontogenic,
Adipogenic, Chondrogenic,
Myogenic, Neurogenic
PDL like formation,
Cementum matrix formation
[32]
BMSC
++++++++
Osteogenic, Chondrogenic,Adipogenic,Neurogenic, Myogenic.
[33]
Human Embryonic
Stem Cells
+++
All three germ layer representatives
[34]
Scaffold Characteristics and Different Biomaterials Used for Tooth Tissue Engineering
Like cell source and type, scaffold is also one of the most important elements in tissue regeneration.
Scaffold provides a physicochemical and biological three-dimensional microenvironment for cell
growth, adhesion, migration and differentiation. Its complex structure and properties guide the cell
to differentiate into specific tissue. To mimic the physiological functions of the natural
extracellular matrix of the cells, scaffold must have to bear some unique characteristics. To address
the biomimetic requirements, a tooth scaffold should have the following characteristics [37, 38, 39].
1. relatively easy to handle;
2. allow for the incorporation of cells and act as a substrate for matrix deposition;
3. provide temporary mechanical support to the affected area;
4. contain a porous architecture to allow for vascularization and tissue-in-growth;
5. allow for the free diffusion of cells and growth factors;
6. permit the establishment of a vascular bed to ensure survival of the implanted cells;
7. should be effective for transport of nutrients, oxygen, and waste;
8. support and promote cell differentiation in the synthetic scaffold;
9. enhance cellular activity towards scaffold-host tissue integration;
10. degrade in a controlled manner to facilitate load transfer to developing tooth;
11. produce non-toxic degradation products;
12. do not incite an active chronic inflammatory response;
13. be capable of sterilization without loss of bioactivity;
14. deliver bioactive molecules or drugs in controlled manner to accelerate healing and prevent
pathology;
15. be ultimately biodegraded and replaced by regenerative tissue, retaining the feature of the
final tissue structure.
Biomaterials used for fabricating scaffold, should be biodegradable and biocompatible. The rate
of biodegradation should be equal to the rate of tissue formation; so that when the tissue is fully
developed the biomaterial will get completely degraded or absorbed in the body. For the scaffold
fabrication, various types of biomaterials, including natural and synthetic polymers, have been
employed (Table 4). Much of the body’s native extracellular matrix is composed of natural

materials like collagen, elastin, fibronectin and laminin. Natural materials, mostly natural
polymers, are cost effective, eco-friendly, have good biodegradability but they have some
disadvantages (Table 4). Some synthetic materials have also been used for tooth tissue engineering
due to flexibility in tailoring the physical, mechanical and chemical properties, and easy
processability of scaffold into desired shape and size. But they too have some disadvantages (Table
4). Therefore, nowadays composite scaffolds (combination of two or more polymers) are used to
overcome the disadvantages associated with the natural and synthetic polymers. Various composite
polymers used for tooth tissue engineering are listed in Table 4.
Table 4. Different scaffold biomaterials employed for tooth tissue engineering along with their
advantages and disadvantages
Scaffold biomaterials
Advantages
Disadvantages
Natural
materials
Bone sialoprotein [40]
Collagen [41]
Fibrin gel [41]
Chitosan [42]
Silk [43]
Platelet-rich plasma [44]
Mineral trioxide aggregate [45]
Cost effective, eco-friendly,
good biodegradability, low
toxicity, low manufacture costs,
low disposal costs and
renewability, have the
properties of biological
signalling, cell adhesion, cell
responsive degradation
Undergoing rapid
degradation, possibility of
losing their biological
properties during scaffold
fabrication processes, the
risk of immunorejection
and disease transmission.
Synthetic
materials
Polylactic acid [46]
Polyglycolic acid [46]
Polycaprolactone [46]
Polylactic-co-glycolic acid [47]
β-Tricalcium phosphate [48]
Hydroxyapatite [49]
Flexibility in tailoring the
physical, mechanical and
chemical properties, and easy
processability of scaffold into
desired shape and size
Adverse tissue reactions
caused by acidic
degradation products, lack
of cellular adhesion and
interaction.
Composite
materials
Glycidyl methacrylated
dextran/gelatin hybrid hydrogel [50]
Polyethyl methacrylate-co-
hydroxyethyl acrylate [48]
Gelatin-chondroitin-hyaluronan tri-
copolymer [51]
Polycaprolactone-gelatin-
hydroxyapatite tricopolymer [52]
More tailorable properties with
the possibility of controlling
biocompatibility, mechanical
properties, tunable degradation
kinetics and physical structures
to suit various applications.
Composite materials
generally don’t have any
disadvantage as they are
the combination of two or
more polymers to impart
various desired properties.
Growth Factors (Morphogens) for Tooth Regeneration
Tooth cannot be considered as a single organ, but somewhere between 3-16 organs, because each
tooth in the upper and lower jaw quadrants is different, making a possible eight different shapes per
jaw. In order to engineer a bio-tooth, an understanding of the molecular principle of tissue
developments and morphogenesis is a prerequisite factor. The promotion and maintenance of
morphogenesis is achieved by a variety of morphogens or growth factors. Growth factors are
naturally occurring proteins capable of stimulating cellular proliferation and differentiation. They
act as signalling molecules that coax the cells to differentiate into specific phenotype. These
signalling molecules help in tooth development and patterning. They also control stem cell activity
by increasing the rate of proliferation, inducing differentiation of the cells into another tissue type,
or stimulating stem cells to synthesize and secrete mineralized matrix. A variety of growth factors
have been successfully used for dentin-pulp complex regeneration, including Transforming Growth
Factors (TGFs) [53], Bone morphogenetic proteins (BMPs) [54], Platelet-derived growth factor
(PDGF) [55], Insulin-like growth factor (IGF) [56], Fibroblast Growth Factors (FGF), Epidermal
Growth Factors (EGF) and phosphates, as discussed in Table 5. According to the origin of stem
cells or cell source, various approaches have been used for tooth tissue regeneration, which are
outlined in Table 6.

Table 5. Signalling molecules, their origin, members, and functions in tooth tissue engineering.
Family
Growth
factor
Member
Primary Origin
Receptors
Functions
Ref.
TGF-β
super-
family
BMP
BMP2- BMP8
Dentine matrix, activated
TH1 cells (T-Helper) and
Natural Killer cells
Transmembrane
serine/threonine
kinase of type I
and type II BMP
receptors
Determine cusps number and position, and therefore the tooth patterning,
required for the transition of tooth development from the bud to the cap
stage, promotes proliferation, differentiation of mesenchymal cells, e.g.,
PDL fibroblasts, follicle cells, fibroblasts, toward a cell with capacity to
form mineral matrix, e.g., osteoblasts and cementoblasts.
[57]
FGF family
FGF
FGF 2,FGF 3,
FGF 4, FGF 8,
FGF 9,FGF 10
A wide range of cells
Notch pathway
Function at distinct steps of odontogenesis, from tooth initiation to the
formation of the last tooth cusp, restrict tooth forming sites by inducing
the expression of Pax9, Pitx1, and Pitx2. FGF10 is able to stimulate cell
proliferation only in the dental epithelium but not in the mesenchyme,
FGF4 is activated by the Wnt signalling pathway, promotes proliferation
and attachment of endothelial and PDL cells.
[58]
EGF family
EGF
EGF, TGF-α
Submaxillary glands
Tyrosine kinase
receptors
Stimulated cell proliferation in tooth explants but at the same time
inhibited tooth morphogenesis and dental cell differentiation.
[59]
IGF family
IGF
IGF-1, IGF-2
IGF-1-Liver
IGF-2 – variety of cells
Tyrosine kinase
receptors
Promotes cell growth, migration, proliferation, differentiation and induce
the synthesis of enamel specific proteins like amelogenin and
ameloblastin.
[60]
PDGF
family
PDGF
PDGFA,
PDGFB,
PDGF C,
PDGF D
Platelets
Tyrosine kinase
receptors
PDGF A regulates the size and stage of tooth development.
[61]
Hedgehog
(Hh) family
Sonic
hedgehog
(Shh)
Shh
Notochord (Central nervous
system)
Patched receptor
Shh signalling is required for the development of early tooth germ
.When Hh signal is decreased below certain degree, the maxillary
incisors are missing.
[62]
Wnt
(Wingless
integrated)
gene family
WNT
Wnt1, Wnt2,
Wnt3,Wnt3a,
Wnt4,Wnt5a,
Wnt 10a,
Wnt 10b
secreted by specific cells to
regulate cellular programs in
the surrounding tissue
Frizzled family of
receptor and low-
density
lipoprotein (LDL)
receptor, known
as LRP5/6.
Play role in determination of the regions of the future tooth formation
and those that will form the remaining of the oral ectoderm.
Detailed function for each Wnt member in tooth development is not
clear at present.
[63]
Transferrin
_
_
Serum protein
_
Necessary growth factor for early tooth morphogenesis, required for the
development of bud- and early cap-staged tooth.
[59]
Emdogain;
Amelogen-
-ins
_
_
Tooth germ
_
Regeneration of periodontal tissues by periodontal cell attachment and
growth, contributes in cementum formation.
[64]
Parathyroid
hormone
(PTH)
_
_
Parathyroid gland
_
Epithelial-mesenchymal interactions during the formation of teeth, helps
in tooth eruption.
[65]
Phosphates
_
_
-
_
Cells within the periodontium are highly sensitive to changes in
phosphate/pyrophosphate levels, it may directly control expression of
genes associated with maturation of cementoblasts and osteoblasts.
[66]

Table 5. Various approaches used for regeneration of dental tissues, according to the source of cells and biomaterials used [67].
Approach
Cell Source
Technique
Biomaterial
Relevance
Ref.
Periodontal
regeneration
Canine (beagle)
Harvest cells, seed onto collagen
sponge, implant against
Periodontium
Collagen sponge (70%Type 1,
30% Type 2)
Potential of in situ tissue engineering using
autologous cells for the regeneration of
periodontal tissues.
[68]
periodontal ligament Stem
cells of miniature pig
Harvest cells, seed onto collagen
sponge, implant against
Periodontium
Hydroxyapatite/ tricalcium
phosphate scaffold
Feasibility of using stem cell-mediated tissue
engineering to treat periodontal diseases.
[69]
Endo-dontal
regeneration
Human stem cells from
exfoliated deciduous tooth
Seed cells onto scaffold and place
in prepared canals of human tooth
D,D-L,L-polylactic acid scaffold
Possible to implant tissue-engineered pulp into
tooth after cleaning and shaping.
[70]
Hard tissue
Apical-pulp-derived
cells, human molar
Harvest of human apical pulp,
expansion in vitro, seed onto
hydroxyapatite scaffold, implant
subcutaneously into nude mice
Porous hydroxyapatite scaffold
The human tooth with an immature apex is an
effective source of cells for hard-tissue
regeneration.
[71]
Scaffold-
based tooth
regeneration
Tooth bud cells, rat pups
Harvest, in vitro expansion, seed
onto scaffold for in vivo
maturation
Porous hexafluoroisopropanol
(HFIP) silk scaffolds
Generation of mineralized tissues for tooth-tissue
engineering; use of silk scaffold.
[72]
Tooth bud cells, rat pups
Harvest, in vitro expansion, seed
onto scaffold for in vivo
maturation
PGA and PLGA scaffold
materials
Tooth-tissue engineering methods can be used to
generate both pig and rat tooth tissues.
[73]
Tooth bud cells, porcine
crown
Harvest, seed onto PGA mesh,
implant into omentum of rat.
PGA fibre mesh
Development of tissue-engineered tooth closely
resembles the pattern of odontogenesis.
[74]
Tooth bud cells, porcine
molar
Harvest, seed tooth cells onto
scaffold, implant into omentum of
rat, join with bone grown in
bioreactor, re-grow in rat.
PGA and PLGA scaffold
materials
Generation of hybrid tooth–bone for the eventual
clinical treatment of tooth loss accompanied by
alveolar bone resorption.
[75]
Organotype-
based tooth
regeneration
Dissociated single cells
from epithelial and mesen-
chymal tissues, recombined
dissociated cells
Harvest of murine tooth bud cells,
implant into tooth socket
Collagen
Proximity of ectodermal and mesenchymal cells
necessary for tooth development; generation of a
structurally correct tooth with penetration of
blood vessels and nerve tissue.
[76]
Tooth bud cells, rat pup
Isolation of tooth bud cells and
co-culture with dental pulp stem
cells, pelletize and culture in renal
capsule
N/A
Mimic the dentinogenic microenvironment from
tooth germ cells in vitro. Demonstrate that
soluble factors can produce a conditioned
medium beneficial for in vitro growth.
[77]
Rat marrow stromal
cells, mouse
embryonic stem cells
mouse embryonic
neural stem cells
Cultured embryonic oral
epithelium with other
mesenchymal cells, transfer tooth
primordium to jaw to grow tooth.
Cell pellet wrapped in epithelium
N/A
Embryonic oral tissue can guide differentiation
of other stem cells to odontoblasts; embryonic
primordium can develop in the adult
environment; generation of a functional tooth.
[78]

Cellular Homing: A Promising Approach for Tooth Regeneration
Stem cell seeding within a scaffold in vitro, was a conventional approach, which aims to mimic
cellular structure and regenerate a functional tissue equivalent in vitro or in vivo. Cellular homing
is an approach which attempts to regenerate tissue in vivo without cell-seeding. Homing is the
process by which cells migrate to and become engrafted in the tissue, on which they exert local,
functional and reparative effects. By using this process, body's own stem cells can be directed
toward the growth factor impregnated scaffolds, made of natural materials, at specific anatomical
sites. Once the stem cells have colonized the scaffold, a tissue can grow and then get merged with
the surrounding tissue. Kim and co-workers used polycaprolactone and hydroxyapatite to fabricate
tooth-shaped scaffold of human molar and rat incisor by applying three-dimensional bio printing
containing 200μm diameter interconnecting micro channels [79]. Two cell homing factors: stromal-
derived factor (SDF)-1 and bone morphogenetic protein (BMP)-7 were infused into the scaffold
micro channels, before implantation. Following mandibular incisor extraction in each of the 22
rats, a rat incisor scaffold was immediately implanted orthotopically, and simultaneously, a human
molar scaffold was implanted ectopically into the dorsum of each rat. The scaffold was colonized
by cells even without exogenously providing cells with the scaffold and for the first time de novo
formation of tooth-like structures and periodontal integration in vivo was found [79]. This strategy
avoids ex vivo cell culture and minimizes the invasiveness associated with clinical procedures. Cell
homing could provide enhancements in cellular methodology for tissue engineering and a novel,
minimally invasive option for tissue regeneration [80]. Because of harnessing the body’s innate
ability to heal and regenerate, it proves to be a meritorious approach in tooth tissue engineering [80,
81, 82]. It is worth mentioning that the tooth tissue regeneration can be achieved by endogenous
regenerative technology that avoids the expense and difficulties associated with culture, storage and
distribution of cells, and immune considerations [82].
Recent Advances in Tooth Tissue Engineering
Partial tooth structures had been regenerated well before the concept of tissue engineering was
introduced by Langer and Vacanti in 1993. In the pulp-capping procedure, dentin production was
known to be induced by calcium hydroxide, although the underlying mechanism was not known
[83]. In 1952, the pioneering work of Shirley Glasstone-Hughes demonstrated that early-stage
embryonic tooth primordia can be split into two, and each half can regenerate a complete normal
size tooth [84]. This established that the early-stage tooth primordia have an inherent level of
plasticity and regenerative capacity. The regenerative capacity of early-stage tooth primordia was
utilized in experiments by Young and co-workers [85], where scaffolds had been fabricated in the
shape of different teeth and cells, dissociated from early-stage third molar tooth germs from pigs
and rats, were seeded onto these scaffolds. The seeded scaffolds were grown in the omentum of
immune compromised rats: histological analysis revealed the formation of tiny (1–2 mm) tooth
crowns 20–30 weeks after the in vivo implantation using porcine tooth buds [85]. The rat molar
tooth germ developed just after 12 weeks of in vivo implantation [86]. It demonstrated the
existence of stem cells in tooth primordia which are able to regenerate teeth. The demonstration of
bioengineered whole tooth crowns from pig and rat tooth bud cells provided the first evidence that
post-natal dental stem cells could be used for whole-tooth tissue engineering applications [85, 86].
Recent studies have indicated that cell-based strategies show promising potential for regenerating
the whole tooth structure in rodents [86, 87, 89]. Moreover, stem cell-based regeneration of human
tooth structures has been achieved in immunocompromised mouse models [90, 91]. One study
indicated that hybrid tooth-bone tissues were bioengineered by seeding the pig third molar tooth
bud cells onto the polyglycolide (PGA) and polyglycolide-co-lactide (PLGA) scaffolds, and
growing for 4 weeks in the omenta of adult rat hosts. There is a tremendous progress in dental stem
cell biotechnology and cell mediated murine tooth regeneration, which encouraged the researchers

to explore the potential for regeneration of living tooth with appropriate functional properties. The
recent advances in tooth tissue engineering are discussed below.
Advances in 2006:
 It was proposed that bone-marrow-stem cells have the potential to turn into both dental
mesenchyme and dental epithelium offering novel possibilities for tooth-tissue engineering
[92].
 Bone marrow mesenchymal stem cells (BMMSCs) were isolated from beagle dogs and
transplanted into experimental Class III periodontal defects. Four weeks after
transplantation the defects were almost regenerated with periodontal tissue. This suggests
that transplanted BMMSCs could survive and differentiate into periodontal tissue cells,
resulting in enhancement of periodontal tissue regeneration. These findings suggest that
auto-transplantation of bone marrow mesenchymal stem cells is a novel option for
periodontal tissue regeneration [93].
Advances in 2007:
 Enamel-dentin complex structures were reconstituted by the cervical loop region cells (CLC)
in recombination with dental pulp horn cell population. In contrast, dentin-cementum
complex structures were reconstituted by cervical loop region cells in recombination with the
dental pulp horn cell population (PHC) within the dental cusp. This shows that cervical loop
cells play a key role in the reconstitution of tooth structure [94].
 It was demonstrated that immortalized mouse dental follicle cells are able to regenerate a
PDL-like tissue in vivo [95].
 It was reported a new technique for regeneration of whole tooth through production of tissue
to replace damaged or missing enamel. Epithelial cells extracted from the developing tooth
of 6-month-old pigs continue to proliferate when they are cultured on top of a special feeder
layer of cells (3T3-J2 cell line). This crucial step boosts the number of dental epithelial cells
available for enamel production and helps in tooth regeneration [96].
 Mesenchymal and epithelial cells were sequentially seeded into a collagen gel drop ex vivo.
After implantation into subrenal capsules in mice, a correct tooth structure comprising
enamel, dentin, root, dental pulp and bone was observed, showing penetration of blood
vessels and nerve fibres [97].
Advances in 2008
 Dentin pulp like tissue had been regenerated when polylactic-co-glycolic acid polymeric
porous scaffolds was seeded with dental pulp stem cells. In this study, it was observed that
the cells formed mineralized-like structures even without the addition of any differentiation
chemicals [98].
 Autologous PDLSCs are capable of forming bone, cementum and periodontal ligament if
transplanted onto hydroxyapatite tricalcium phosphate (HA/TCP) carrier into periodontal
defects created surgically. For periodontal regeneration, non-dental derived stem cells have
been tested. ASCs (Adipose-derived Stem Cells) isolated from rat and mixed with platelet-
rich plasma (PRP), were implanted into the periodontal tissue defect generated in the test rat.
Partial alveolar bone regeneration and a periodontal ligament-like structure were observed 8
weeks after implantation [99].
 Human exfoliated deciduous teeth (SHED) constitute a viable source of stem cells for dental
pulp tissue engineering. A new dental pulp-like tissue was engineered, when SHED was
seeded onto dentine slices and implanted subcutaneously in immunodeficient mice. It was
also demonstrated that SHED when co-implanted with human endothelial cells, improve the

newly formed tissue-organization, microvascular network, and oxygen and nutrient influx.
It was shown that the resulting tissue presented architecture and cellularity that closely
resemble those of a physiologic dental pulp [100].
 For biological tooth to perform adequately, they must be able to grow and integrate into the
existing alveolar bone of the jaws. Amazingly, implanted embryonic tooth germs can
develop normally and form tooth when transplanted into the adult mouse. Further analyses
using 3D imaging have shown that the tooth not only have normal morphology, but also can
erupt into the oral cavity and form functional roots [16].
 A viable biological substitute of a tooth was engineered by seeding the tooth shaped scaffold
with dental germs and placing it in rat jaw or omentum [101]. It has been demonstrated that
a full tooth can be produced by using the techniques of tissue engineering as illustrated in
Figure 5.
Figure 5. Schematic showing the process of bioengineering dental tissue which is grown in the
omentum or rat jaw.
Advances in 2009
 The hybrid tooth-bone construct was prepared by combining tooth-bud cell seeded scaffolds
with autologous iliac-crest bone-marrow-derived stem cell seeded scaffolds, which were
transplanted into surgically created mandibular defects in the minipig. It was found small
tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal
ligament, surrounded by regenerated alveolar bone. This study suggests the possibility for
regeneration of teeth and associated alveolar bone, in a single procedure [102].
 Evidence for engineering tooth enamel has been found that will help people to replace lost
parts of the tooth with a healthy layer of new enamel. A simple amino acid, proline, which
is repeated in the centre of proteins found in tooth enamel, makes teeth stronger and more
resilient. When the repeats are short, such as in frogs, teeth will not have the enamel prisms
that are responsible for the strength of human enamel. In contrast, when the proline repeats
are long such as in human being, they contact groups of molecules that help enamel crystals
grow [103].
Advances in 2010
 Use of heterogeneous population of stem cell-mediated de novo regeneration of pulp-dentin
tissue has been demonstrated. It was found that pulp-like tissue can be regenerated de novo
in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that
give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.
Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated,
characterized, seeded onto synthetic scaffolds consisting of poly-D, L-lactide/glycolide,

inserted into the tooth fragments, and transplanted into mice. Continuous layer of dentin
like tissue has been shown to be formed on the canal dentinal walls (Figure 6) and on
mineral trioxide aggregate (MTA) cement surface [104].
Figure 6. SCID mouse as subcutaneous study model for pulp-dentin regeneration. The canal space
of human tooth root fragments (*6–7 mm long) was enlarged to *1–2.5 mm in diameter. One end
of the canal opening was sealed with MTA cement (SCID, severe combined immunodeficient;
MTA, mineral trioxide aggregate) (reproduced with permission from reference 104).
 It was shown that in mice the Jagged2 gene is required for the healthy development of teeth.
Inactivation of this gene interrupts the Notch signalling pathway and due to this tooth
crowns will be malformed (Figure 7) and enamel will be lacking [105].
Figure 7, Shape of a healthy (left) and deformed molar with the Jagged2-gene deactivated (right) in
the mouse embryo (reproduced with permission from reference 105).
Advances in 2011
 Recently, there is a finding which assures great promise for regenerative medicine. Many
types of cells including DPSCs, SHEDs, and SCAPs have been successfully reprogrammed
into induced Pluripotent stem (iPS) cells. Autologous DPSCs are isolated from the patient’s
own dental pulps and pluripotent iPS cells are created by driving four genes (c-Myc, Klf4,
Oct3/4, and Sox2) into DPSCs, which can be used to generate dental epithelial cells under
suitable conditions. These iPS-derived dental epithelial cells are recombined with
autologous DPSCs pellet and transplanted in vivo to make a bio-tooth. After the temporary

incubation in vivo, the bio-tooth can be transplanted into the patient’s jaws to treat tooth
loss. These dental iPS cells express the marker genes that characterize embryonic stem
cells, and maintain the developmental potential to differentiate into advanced derivatives of
all three primary germ layer [106, 107].
 Takashi Tsuji and his co-workers [108] isolated stem cells from the molar teeth of mice, and
these stem cells were placed in a mold to grow. When the full tooth units were grown, it
was transplanted into the jaws of one month old mice. After 40 days, it was observed that
the transplanted teeth get fused with the jaw bones, and nerve fibres were also present in the
growing teeth. The mice that received the transplanted teeth were able to eat and chew
normally without any complications.
Challenges and Future Prospective in Tooth Tissue Regeneration
The term “tissue engineering” is now almost 35 years old, and significant progress has been made
in tooth tissue engineering during the last decades. But still, tissue engineering has not yet matured
enough into such a level so that the engineered tooth is widely accepted as autograft or allograft for
dental treatments. The regeneration of a whole tooth structure including enamel, dentin/pulp
complex, and periodontal tissues as a functional unit in humans is a challenge with the currently
available regenerative biotechnologies. It has been accepted that formation of multiple miniature
tooth crowns in the bioengineered tooth constructs is possible, but real-size whole-tooth
regeneration encounters a number of challenges due to the complexity of human tooth growth and
development. Therefore, there are still significant hurdles to overcome in developing tissue
engineered tooth and to bring it out successfully from the laboratory to clinical use. Some of the
multifold challenges, as described below, must be surmounted for rapid progress in tooth tissue
engineering.
1. Source of stem cells - Stem cells can be obtained from any source, autogenic or allogenic.
Although the autogenic stem cells have no risk of immune rejection, are least expensive and
have no ethical concerns, but to use them is time consuming procedure. Using allogenic
stem cells saves considerable time but the risk of immune rejection and pathogen
transmission limits their use. Xenogenic tooth bud cells may lead to dysmorphogenesis of
regenerated teeth, even if it is applicable to humans. Isolation, culture and storage of these
stem cells are technique-sensitive, and their immunomodulatory characteristics are still
questionable.
2. Non-vital nature of enamel - Enamel is fundamental to oral function, and yet it is
frequently the first to be lost. Innovative solutions are required to compensate for significant
loss of enamel.
3. Control of further development within the body - As the tissue engineered parts have
been developed using very potent signal molecules to induce the alteration of the growth of
stem cells, a way has to be found to insure that these alteration and growth will not continue
beyond control when implanted.
4. Implantation of the tissue engineered parts - From the stage of fabrication to the stage of
implantation, these tissue-parts have to be kept alive, and they have to find a functional
place within the human body, with a proper supply of blood, oxygen and nutrients. In
dentistry, implanting a tissue engineered tooth would be a very difficult task, when contact
with the bone, size, shape and positions of the implanted tooth are so important to a proper
and functional implantation.
5. Aging - One of the obstacles is to match the aging of the implanted tissue engineered parts
with that of the surrounding tissues and organs.

6. Cost - tissue engineered products should be economical so that the patient can afford the
treatment.
7. Storage - Cryopreservation or hypothermic storage of tissue-engineered tooth tissues should
be made so that when needed it is easily available.
8. Issues involved in the reconstruction of a bio-tooth - shape determination, tooth size
(engineered tooth is smaller than that of normal) availability of dental epithelium, directional
growth and eruption, spatial organized pulp, dental and enamel, mechanical strength and
graft rejection in the jaws remain to be resolved.
Conclusions
A new discipline in dentistry, ‘regenerative dentistry’, has been resulted due to recent advancements
in scaffold designing, better understanding of growth factor biology and interactions between
allogenic stem cells and immune system. It provides an increased level of understanding of the
mechanical and chemical stimuli that regulate tissue responses. Tissue engineering certainly has the
advantage over the other options in its low cost, small recovery time, non-intrusiveness, and as a
permanent fix to the problem. With the studies and development to come in the future, it will be
able to prove to be the most effective and long lasting procedures. The accumulating knowledge on
tooth tissue engineering can be applied to regenerate missing osseous or dental structures, repair
and replace mineralized tissues, promote oral wound healing, correct orofacial deformities and the
periodontal surgery. Biomechanical principles can also be applied to tissue engineering to enhance
the bone/tooth or bone/implant functionality and long-term stability. Dental tissue-engineering
companies, such as Dentigenix and Odontis, and large-scale enterprises such as Hitachi, have
greatly accelerated the research process and have also introduced the commercial products. Several
researches are going on mice but upscaling to humans, appears to be highly challenging.
Advancements are also being achieved in the area of biomimetics that will allow the creation of
new biologic replacements for missing oral structures. Several advancements have been made but
still improvement is necessary in the success rate of tooth formation and in the morphology of
regenerated tooth in tissue engineering methods. The opportunity for bioengineering to employ the
course of tooth regeneration is an exciting prospect and will improve the quality of life for patients
for decades to come. Tissue engineering does not end here, but will have a future in many aspects
of the medical field and each of our lives.
Acknowledgements
The authors are grateful to the Council of Scientific and Educational Research (Grant No.
27(0222)/1 O/EM R-II dated 31.05.10), India, for funding this work.
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