Tooth development proceeds with reciprocal inductive interactions between stomadeum ectoderm and underlying ectomesenchymal cells in a strictly controlled temporal and spatial order. Well studied at the molecular biologic level, over 300 genes and 100 growth and differentiation factors are implicated in the control of cellular differentiation and crosstalk in dental development that result in structures containing combination of mineralized tissues (enamel, dentine, cementum), soft connective tissues (dental pulp, periodontal ligament), blood vessels, nerves and lymphatics

2. Teeth Regeneration
By
Romissaa Aly Esmail
• Assistant lecturer of Oral Medicine, Periodontology,
Diagnosis and Dental Radiology (Al-Azhar University)

3. Content:
• Development of Teeth—Complex Multi-tissue
Structures
• Regeneration of Dental Tissues and Presence of
Resident Stem Cells
• Periodontal/Endodontic Tissue Engineering Versus
Whole Tooth Germ Tissue Engineering

5. • Tooth development proceeds with reciprocal
inductive interactions between stomadeum
ectoderm and underlying ectomesenchymal cells
in a strictly controlled temporal and spatial order.

6. • Well studied at the molecular biologic level, over 300 genes and
100 growth and differentiation factors are implicated in the control
of cellular differentiation and crosstalk in dental development that
result in structures containing combination of mineralized tissues
(enamel, dentine, cementum), soft connective tissues (dental
pulp, periodontal ligament), blood vessels, nerves and lymphatics.

8. The analysis of the signalling
pathways reveals an
interdependent role of both
the epithelium and
mesenchymal tissues, with
the initial induction capability
residing in the epithelium

9. Prior to the arrival of cranial neural crest
cells into the first branchial arch, the
endodermal stimulus results with the
regionalization of the oral ectoderm into
proximal (molar, posterior) and distal
(incisor, anterior) domains
which are characterized with the differential
expression of growth factors (fibroblast
growth factors-FGFs, bone morphogenetic
proteins-BMPs) and signalling molecules
(Sonic hedgehog-SHH, Wingless INT genes-
WNTs)

10. Thus, while the FGF8 is
predominantly expressed in
proximal domain,
BMP-4 is prevalent in the distal
one, and the concentration of the
signalling molecules determine the
fate of the neural crest cells that
will populate the area and form
ectomesenchymal condensation.

11. The FGF8 and BMP-4 inhibit each other and subsequently
appear to be expressed in both ectoderm and ectomesenchyme,
while SHH and WNTs expression remains restricted to the
ectoderm.
SHH and WNTs play a role in determination of the regions of
the future tooth formation and those that will form the remaining
of the oral ectoderm.
Once the regions of tooth formation in oral ectoderm are
determined, the molecular interactions trigger the formation of
shoe-like ectodermal area that will correspond to the future
dental arches called the dental lamina
Increased cellular proliferation areas on the dental lamina result
with the epithelial thickenings, the dental placodes, which will
subsequently invaginate into the underlying ectomesenchymal
tissue that also condenses beneath them, and form tooth buds

12. The tooth buds further develop
inside the ectomesenchymal tissue
with continuous reiterative signalling
between the two compartments
determining the precise
morphogenetic outcome during
mammalian dental organogenesis .
The tooth bud undergoes transition
through cap and bell stages that finally
results with the formation of the germs of
primary teeth in humans.
The secondary tooth germs develop
from the small diverticulums springing
from the superficial side of the tooth buds,
i.e. epithelial tissue that connects the bud
to the oral ectoderm.
The primary tooth germs in humans begin
to develop from the 6th week of gestation
and takes the form of a cap by the 10th
week, proceed as a bell and erupt in early
childhood, while the secondary germs
pause at the bell stage until the
replacement of the primary teeth in
children.

14. Of all the dental structures, only enamel is incapable of regenerating its original
structure, while the remaining tissues possess that capacity in varying degrees,
dependent on multiple factors

15. Thus, the ability of
restoring the damaged
tissue structure is a
result of cellular
activities in a time- and
localizationdependent
fashion, involving
proliferation, migration,
extracellular matrix
synthesis and secretion
and molecular
mechanisms regulating
intercellular interactions.
The presence of blood
flow appears to play a
pivotal role, ensuring
nutrient and oxygen
supply, as well as
metabolite removal in the
sites of cellular activity.
Both pulpal and
periodontal tissues are
highly vascularised,
supporting the progenitor
cell activities following
tissue damage and injury.

16. The stimuli resulting from
bacterial attack, mechanical and
thermal trauma, and
inflammatory processes in the
pulpal tissue triggers the
reparative response of the
odontosblastic cells lining the
dentinal surface of pulp
chamber, which results with the
increased cellular proliferation
and extracellular matrix
synthesis and deposition of
reparative (reactionary) dentin

17. The processes of reparative dentinogenesis
and the inflammatory reaction of the pulpal
tissue are interdependent, with the milder
form of the latter triggering odontoblast
progenitor and stem cell activation .
The presence of stem cells in both deciduous and permanent teeth
pulp tissue is demonstrated and their proliferation, differentiation
and stem cell properties were extensively characterized.

18. Indeed, multipotent adult
stem cells exhibiting common
embryonic stem cell
transcription factors
such as Oct4 and Nanog have
been isolated from adult bone
marrow, neural tissue,
connective tissue of oral
mucosa, peripheral blood [54],
skeletal tendons, adipose
tissue, hair follicle, and so on.

19. Such a possibility may cast doubt on the uniformity of periodontal
ligament stem cells, and multiple cell subsets possessing stem cell
characteristics but with different tissues-generating capabilities could be
isolated depending on the techniques and characterization methodology.

21. Stem cells can continuously
produce unaltered
daughters and could
generate cells with different
and more restricted
properties.
Stem cells can divide either
symmetrically (allowing the
increase of stem cell
number) or asymmetrically.
Asymmetric divisions keep
the number of stem cells
unaltered and are
responsible for the
generation of cells with
different properties.
These cells can either
multiply (progenitors or
transit amplifying cells) or be
committed to terminal
differentiation

22. • Embryonic stem cells are pluripotent, i.e. can differentiate into
representatives of all three germ layers, all the somatic cells
except the germ cells.
Their differentiation potential is
more restricted only from the
totipotent zygote and the cells of
the morula, which can form the
complete organism in mammals .
Stem cells from adult tissues are
considered multipotent at best,
with the putative ability of
transdifferentiation remaining an
intensely contested issue .

26. Intercellular communication
pathways within the Basic
Multicellular Unit (BMU) that
comprise the remodeling
process in trabecular bone.
(1) Stimulatory and
inhibitory signals from
osteocytes to osteoblasts
(e.g., OSM, PTHrP, and
sclerostin).
(2) Stimulatory and
inhibitory signals from
osteoclasts to osteoblasts
(e.g., matrix-derived TGFβ
and IGF-1, secreted CT-1,
Sema4D, and S1P).
(3) Signaling within the
osteoblast lineage (e.g.,
ephrinB2 and EphB4,
Sema3a, PTHrP, OSM).
(4) Stimulatory and
inhibitory signals between
the osteoblast and
osteoclast lineages (e.g.,
RANKL, Sema3B, Wnt5a,
and OPG).
(5) Marrow cell signals to
osteoblasts (e.g.,
macrophage-derived OSM,
T-cell-derived interleukins,
and RANKL).
Hemopoietic precursors
(HSC), bone mesenchymal
stem cells (MSCs

27. The realization that implanted stem cells of adult or embryonic origin integrates
into the host neural tissues and triggers the regenerative response with improved
function as a result of trophic action and stimulation of the resident populations,
but not conversion into neural cells underscores this shift of paradigm

28. The generation of whole tooth germ
for implantation however would
require complete recapitulation of
the odontogenic developmental
program, involving epithelial and
ectomesenchymal cells competent
in odontogenesis, with the resultant
structures capable to integrate with
the host vasculature and nervous
system

29. Thus far, no adult cells with the ability to initiate
odontogenic program like the stomadeum
ectoderm have been identified
Although disintegrated cells from impacted third
molars were able to reorganize in biomaterial
scaffolds to form teethlike structures, gross
anatomical deficiencies indicate that such an
approach remains insufficient to achieve
functional tooth replacement .

30. However, the periodontal and pulpal tissues are
derived from ectomesenchyme, a neural crest cells-
derived tissue different in origin from the mesodermal
tissues.
This may explain the failure of
inducing mesodermal cells along
the odontogenic program to
complete normal tooth structures
even under the inductive influence
of dental ectoderm.

31. Recombination experiments with embryonic tissues or those utilizing adult
dental stem cells and odontogenically competent inductive epithelium
demonstrate the feasibility to recapitulate the odontogenic developmental
program resulting with the formation of complete tooth germs.

32. • The difficulty is associated with the maintaining of controlled
culture conditions for several months, where minor fluctuations in
the environmental variables could unpredictably shift the
molecular interactions at cellular level, resulting with hundreds of
different morphological outcomes

33. Periodontal/Endodontic Tissue
Engineering Versus Whole Tooth
Germ Tissue Engineering

34. Contemporary techniques
allow only for partial
regeneration of lost
periodontal structures
with variable degree of
success depending on
multiple factors, and also
limited pulpal regeneration
in relatively minor
degenerations.

35. Several clinical applications
performed as pilot
controlled studies report
the successful utilization of
autologous gingival cells for
mucogingival and root
coverage procedures.
. Autologous dental pulp
stem cell seeded
collagen sponges were
effective in the
regeneration of alveolar
bone defects distal to
second molar teeth
following third molar
extraction.

36. Cultured periosteal cells also
contributed to enhanced
regenerative outcome in
infrabony periodontal defects
when combined with platelet-rich
plasma and porous
hydroxyapatite granules .
However, the treatment applications based on autologous cell
implantation require stringent good manufacturing practice
(GMP) standards and clinical efficacy is still variable at best

44. Isolation, Culturing, and Seeding of Rat Tooth Bud Cells Molar tooth buds were
isolated from 3- to 7-dpn Lewis rat pups and minced into < 1-mm-sized pieces in
37°C Hanks' balanced salt solution (HBSS, Gibco BRL, Gaithersburg, MD, USA).
Tooth bud tissues were digested with type I collagenase (0.66 mg/mL,
SigmaAldrich, St. Louis, MO, USA) and Dispase I (0.33 mg/mL; Boehringer
Mannheim, Indianapolis, IN, USA), dissociated by trituration,
and washed 5x in 50% Dulbecco's modified Eagle medium (DMEM, Gibco BRL,
Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS), 5 mL Glutamax,
50 units/mL penicillin, 50 g/mL streptomycin, 2.5 g/mL ascorbic acid, and 50% F12
medium (Sigma-Aldrich Corp, St. Louis, MO, USA).
Single-cell suspensions were generated by filtration through a Falcon 40-micron cell
strainer, typically yielding 2.4 x 105 cells/tooth bud.

45. Cells were resuspended in DMEM/F12, plated into
75-cm2 (T75) culture flasks (Costar, Cambridge, MA,
USA) at 2.5 x 105 cells/mL, and grown in 5% CO2 at
37°C until the cells reached confluence at 6 days.

46. Immunohistochemical analysis
of cytokeratin expression in
cultured epithelial tooth bud
cells was performed with the
use of the monoclonal pan-
cytokeratin antibody PCK-26
(Sigma-Aldrich, St. Louis, MO,
USA), according to the
manufacturer's recommended
protocol.
Cells were harvested by
trypsinization (0.25%
trypsin/EDTA; GibcoInvitrogen
Corp., Tulsa, OK, USA) for 10 min
at 37°C, washed twice with the
same medium, recounted, split
into equal portions, and
statically seeded onto PGA and
PLGA scaffolds for 1 hr prior to
implantation into the omenta of
syngeneic Lewis rat hosts.

50. Figure 7 The therapeutic
effect of DPSC-EXO on
periodontitis in rats. (A)
Representative sagittal
3D and 2D views of the
maxillary molars from
micro-CT scanning.

51. The red line corresponds to
the distances of ABC-CEJ.
(B) and (C) Measurement of
the BV/TV and the distance
from the ABC to the CEJ in
each group.
(D) Representative H&E-
stained sections of
periodontal tissues
harvested from each group
at low and high
magnifications.
The green box shows the
details of gingiva, and the
yellow box shows the
details of alveolar bone
crest.
(E) The degree of
osteoclast infiltration in
representative TRAP-
stained sections of
periodontal tissues.
(F) Quantitative analysis of
osteoclast number
between the first and
second molars.
The yellow arrows show
that osteoclasts are
stained red.
(B) Buccal side; (P) Palatal side;
PD: Periodontitis; BV/TV: Bone
tissue volume/tissue volume
(%); ABC-CEJ: Alveolar bone crest
and the cementoenamel
junction; Results are the mean ±
s.d. (*p < 0.05, **p < 0.01, ***p