This document discusses stem cells, providing a historical background of stem cell discoveries from 1908 to present. It defines stem cells and categorizes them into embryonic, adult, and induced pluripotent stem cells. Various sources of adult stem cells are described, including bone marrow-derived mesenchymal stem cells and different dental tissue-derived stem cells like dental pulp stem cells, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle stem cells. Studies on the potential of these stem cells for periodontal regeneration are summarized.

1. STEM CELLS
1
“Pro Life Paves Path For Life”
Dr.R. DHIVYA .,MDS

2. CONTENTS
 Introduction
 Historical background
 Def and types of stem cells
 Source and derivation of various stem cells
 Individual stem cells
2
 Stem cell markers
 Role of stem cells in periodontics
 Challenges encountered
 Future directions
 Conclusion

3. INTRODUCTION
3

4. HISTORICAL BACKGROUND
4
Year Discoveries
1908 Alexander Maksimov propose the term "Stem Cells - were later revealed to be mesenchymal stem cells.
1960 Two scientists, Joseph Altman and Gopal Das, present scientific data that indicate the existence of neural stem cells
1963 James Edgar Till, along with Ernest McCulloch, are the first to illustrate the existence of hematopoietic stem cells
1968 A bone marrow transplant is successfully used between two siblings for the treatment of (SCID).
1981 Martin Evans along with Matthew Kaufman, manage to extract mice embryonic stem cells from mice blastocysts.
1992 Brent A. Reynolds and Samuel Weiss manage to isolate neural stem cells from the adult striatal tissue,
2001 Researchers of Advanced Cell Technology become the first ones to clone early staged human embryos
2004 Hwang Woo-Suk announced the creation of several human embryonic stem cell lines from unfertilised human oocytes.
2007 Shinya Yamanaka again comes first. This time, for being the first one to create human induced pluripotent stem cells.
2010 The first human clinical trial involving embryonic stem cells commences.
2012 Advance Cell Technology announces human stem cell clinical trial

5. 5

6. 6
Three basic categories of cells make-up the human body:
 Somatic cells include the bulk of the cells that make-up the human
adult and each of these cells in its differentiated state has its own copy
or copies of the genome.
 Germ cells are cells that give rise to gametes, i.e. eggs and sperm.
 Stem cell is a cell with the ability to divide indefinitely in culture
and with the potential to give rise to mature specialized cell types.

7. DEFINITION
 A ‘‘stem cell’’ refers to "a clonogenic, undifferentiated cell that is
capable of self-renewal and multi-lineage differentiation". -
(Smith A. A glossary for stem-cell biology -1981)
 In other words, a stem cell is capable of propagating and generating
additional stem cells, while some of its progeny can differentiate and
commit to maturation along multiple lineages giving rise to a range of
specialized cell types.
 Depending on intrinsic signals modulated by extrinsic factors in the stem
cell niche, these cells may either undergo prolonged self-renewal or
differentiation 7
Stem cell
Stem cell
( unlimited cell
division )
Specialized cell
(e.g., white blood cell)

8. THESE ESSENTIALATTRIBUTES OF ‘STEMNESS’ARE PROPOSED TO INCLUDE:
 The capacity to sense growth factors and interaction with the extracellular matrix via integrins;
 Engagement in the cell cycle, either arrested in G1 or cycling;
 A high resistance to stress with upregulated DNA repair, protein folding, ubiquitination and detoxifier systems;
 A remodeled chromatin, acted upon by DNA helicases, DNA methylases and histone deacetylases; and
Translation regulated by RNA helicases
 Active Janus kinase signal transducers and activators of transcription, TGFb and Notch signalling;[ DNA
transcription – signalling]
8
Ramalho-Santos M, Yoon S, Matsuzaki Yet al. ‘Stemness’: transcriptional profiling of embryonic and adult
stem cells. Science 2002; 298: 597–600.

9. TYPES OF STEM CELLS
9
STEM CELL CATEGORY DEFINITION EXAMPLE
TOTIPOTENT
The capacity to differentiate into all possible cell types
including extra embyonic tissues
Fertilized egg
PLEURIPOTENT
The ability to differentiate into almost all cell type.
Pleuipotent cells lack the capacity to contribute to extra
embryonic tissue and therefore cannot develop into fetal or
an adult animal
Embryonic stem cells
MULTIPOTENT
The potential to give rise to multiple cells,but limited
amount of lineages
Mesenchymal stem cells
OLIGOPOTENT
The capacity to differentiates into few cell type Myeloid stem cells
UNIPOTENT
The ability to differentiate into only one type of cell Skin

10. This cell
Can form the
Embryo and placenta
This cell
Can just form the
embryo
Totipotent
Pleuripotent
Multi-potent

11. 11
According to their origin and differentiation
potential, stem cells are classified as:
 Embryonic stem cells
 Adult stem cells
 Induced pluripotent stem cells

12. Stem cells
Embryonal Stem
Cells (ESCs)
Adult Stem
Cells (ASCs)
Induced Pluripotent
Stem Cells(IPSCs)
Germ Line
Spermatogonia Oogonia
Somatic
pancreas
Liver
intestine
mesenchymal
skin
Hemopoietic
Bone Marrow Dental Pulp
neuronal

13. 13
 Embryonic stem (ES) cells are taken from inside the blastocyst, a very early stage embryo.
 The blastocyst is a ball of about 50-100 cells and it is made up of an outer layer of cells, a fluid-filled space and a
group of cells called the inner cell mass. ES cells are found in the inner cell mass.
Embryonic stem cells

14. 14
 They are capable of giving rise to cells of all
three germ layers.
 They are responsible for the formation of an
individual cell from an embryo, and they disappear
by the time adulthood is attained.
 Hence embryonic stem cells are the best source
of cells for periodontal regeneration due to their
high plueripotency.

15. EMBRYONIC STEM CELLS
15
BLASTOCYST
outer layer of cells = ‘trophectoderm’
cells inside = ‘inner cell mass’
embryonic stem cells taken from
the inner cell mass
culture in the lab
to grow more cells
PLURIPOTENT
fluid with nutrients
all possible types of specialized cells
differentiation
liver
blood
neurons
skin

16.  Embryonic stem cells are ideal for periodontal regeneration.
 However, their use in clinical therapy has been hampered by ethical concerns .
 Another important disadvantage is that, their implantation in the human body has been associated with the
occurrence of rare cancers .
Related studies :
 A bioengineered tooth was developed by Nakao, et al. using murine embryonic stem cells derived from
epithelium and mesenchyme, which was able to erupt from the oral cavity of the mouse and develop into a fully
functional tooth.
16

17. ADULT STEM CELLS
 Adult stem cells are found in the human body and in umbilical cord blood.
 The most well known source of adult stem cells in the body is bone marrow but they are also found in many organs and
tissues; even in the blood.
 Adult stem cells are more specialized since they are assigned to a specific cell family such as blood cells, nerve cells, etc.
17
MULTIPOTENT
blood stem cell
found in
bone marrow
differentiation
only specialized types of blood cell:
red blood cells, white blood cells, platelets

18. ADULT STEM CELLS
 Recently, it was discovered that an adult stem
cell from one tissue may act as a stem cell for
another tissue, i.e. blood to neural
 If they are derived from bone marrow it is called
Bone Marrow derived Mesenchymal Stem
Cells – BMMSC and if derived from tooth –
Dental Mesenchymal stem cell – DMSC .
18

19. BONE MARROW DERIVED MESENCHYMAL STEM CELLS (BMMSC)
19
 The main source of adult stem cells is the bone marrow in
proportion of 1 in 34,000 of nucleated cells.
 MSCs derived from the bone marrow are referred to as bone
marrow derived mesenchymal stem cell (BMMSC) and have
been the most studied amongst mesenchymal stem cells
 BMMSCs have been found to be capable of differentiating
into various cell lineages like osteoblasts, chrondocytes and
adipocytes

20. 20

21.  A study done by KAWAGUCHI et al in 2005 suggest that auto-transplantation of bone marrow mesenchymal
stem cells is novel option for periodontal tissue regeneration. Class III furcation defects were regenerated
with cementum, periodontal ligament and alveolar bone in beagle dogs with MSC – Atelocollagen groups when
compared with control group by placing atelocollagen alone.
 A study done by PIERRI et al in 2009 tested the effect of the combination of mesenchymal stem cells (MSCs)
and platelet-rich plasma (PRP) incorporated into a fluorohydroxyapatite (FHA) scaffold on bone
regeneration in cylindrical defects in the edentulous mandibular ridge of minipigs which shows enhanced bone
formation after 3 months.
21

22. 22
 MSCs has been found in various dental
tissues.
 These are easier to harvest and associated
with lesser patient related complications.
Dental MSCs

23. STEM CELLS OF DENTAL TISSUE ORIGIN
23
Dental pulp stem cells (DPSCs)
Periodontal Ligament stem cell (PDLSC)
Stem cells from apical papilla (SCAP)
Dental Follicle Stem Cells (DFSC)
Stem cells from Human Exfoliated Deciduous teeth (SHED)
Gingival Mesenchymal stem cells (GMSC)
Epithelial Cell Rests of Malassez (ECRM)

24. DENTAL PULP STEM CELLS (DPSCS)
 Carinci and coworkers isolated a subpopulation within the DPSC
with osteogenic potential
 The osteoblasts obtained from these DPSC were compared with
normal osteoblasts and numerous variations were found which
might be responsible for the difference in bony tissue produced
by these cells.
 Also the effect of these stem cells on periodontal regeneration has
been inconsistent based on numerous studies done in beagle dogs
till date .
 In vitro characterization reveals mesenchymal stem cell markers
such as STRO-1, CD31 and CD146 and also embryonic stem cell
markers Oct-4 and Nanog. They also express a mesenchymal
marker vimentin. 24

25. Advantage Of Dental Pulp Stem Cells :
1. DPSC could regenerate a dentin-pulp-like complex, which is composed of mineralized
matrix with tubules lined with odontoblasts, and fibrous tissue containing blood vessels in
an arrangement similar to the dentin-pulp complex found in normal human.
2. DPSC posses striking features of self-renewal capability and multilineage differentiation by
finding that DPSC were capable of forming ectopic dentin and associated pulp tissue in-vivo
and differentiating into adipocytes and neural-like cells.
Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo
S. Gronthos
PNAS:December 5, 2000 vol. 97

26.  A study done by Gronthos et al in 2000 revealed when DPSCs were transplanted into
immuno- compromised mice, they generated a dentin-like structure lined with human
odontoblast-like cells that surrounded a pulp-like interstitial tissue. Thus human DPSCs
would also be capable of regenerating a dentin/pulp-like structure in vivo.
 A study done by Backely et al in 2008 hypothesised the use of poly(lactic-co-glycolic acid)
(PLG) scaffolds with different porosities co-cultured with rabbit dental pulp stem cells
(RDPScs) and transplanting them subcutaneously to evaluate the potential of this stem
cell/scaffold construct to regenerate dentine/pulp tissue.
26

27. PERIODONTAL LIGAMENT STEM CELL (PDLSC):
 Periodontal ligament is a specialized connective tissue that connects
cementum and alveolar bone, to maintain and hence support the teeth in
sight also preserve tissue homeostasis.
 Multipotent stem cells from human periodontal ligament were isolated for
the first time by Seo, et al. He reported that PDLSCs exhibited some
characteristic features similar to BMMSCs .
 The peculiar features were multipotency, clonogenic ability, high
proliferation and expression of putative stem cell marker such as STRO-1
and perivascular cell marker CD 146 .
 Like the BMMSCs they also express CD44, CD90, CD105, CD166.
27

28.  “Scleraxis” which is a transcription factor specific to tendon was found to be highly
expressed by PDLSCs as compared to BMMSCs and DPSCs .
 Hence it was concluded that periodontal ligament derived MSC are one of the most
effective source for periodontal regeneration.
 Park JY et al in 2011 carried out a study in beagle dogs comparing PDLSCs with
stem cells obtained from other dental sources and concluded that PDLSCs are most
predictable in carrying out regeneration.
 Han et al. in 2014 created fenestration defects in murine models and placed
allogeneic PDLSC to assess the time required for the defect to be mineralized. They
observed that by day 14 and 21 significant amounts of mineralized tissue and bony
bridge was formed.
28

29.  Ji, et al in 2010 investigated the effect of PDLSCs derived from retained deciduous teeth (DePDLSC) in
periodontal regeneration and compared it with PDLSCs derived from permanent teeth (PePDLSC).
 DePDLSCs were found to be comparatively immature hence readily differentiated into osteoblasts in
osteogenic medium. Also, they were found to have the higher colony forming ability and increased
proliferation rate as compared to PePDLSCs.
 Further DePDLSC cell sheets when combined with dentin blocks resulted in the formation of PDL and
cementum like tissue on the dentin block however PePDLSCs resulted in formation of no cementum.
 Numerous other studies carried out in periodontal defect models in animals have reported positive results
with application of PDLSC.
29

30. STEM CELLS FROM APICAL PAPILLA (SCAP):
 Apical papilla is the soft tissue present at the apices of developing roots
of permanent teeth.
 It is responsible for the formation of the radicular pulp hence SCAP
resemble DPSCs however, they are comparatively more immature
hence superior for tissue regeneration .
 They are isolated from tips of developing roots, hence can be
harvested easily during extraction of impacted third molars.
30

31.  In vitro characterization reveals MSC markers STRO-1, CD146 and CD24
which seem to be a unique feature of these cells .
 A study done by Sonayama et al in 2008 says that when SCAP have been
incorporated along with periodontal ligament stem cells in extraction
sockets of miniature pigs, resulting in the successful formation of root and
supporting periodontal structures
 They have been considered crucial in root formation which might be
partly because, SCAP are the source of primary odontoblasts responsible
for formation of root dentin
31

32. DENTAL FOLLICLE STEM CELLS (DFSC):
 Dental follicle is an ectomesenchyme derived loose connective tissue sac
surrounding the developing tooth bud from which arises the alveolar bone,
cementum and periodontal ligament.
 DFSCs are relatively easy to harvest as can be procured from the follicles of
unerupted third molars.
 In vitro characterization reveals stem cell markers Nestin, Notch-1 and
STRO-1
 They express vimentin (mesenchymal marker) and cementoblast markers
(Cementum protein-23, Cementum attachment protein).
32

33. 33
A study done by Hilken et al in 2013 suggested that SCAP could potentially promote the vascularization of
regenerated dental tissues both INV ITRO and INVIVO .
 Guo, et al. has reported that DFSCs have the potential for regenerating the entire root of the teeth.
 In support of the role of DFSCs in forming various periodontal structures, the authors discovered a crucial role
played by Wnt5a
 Wnt5a proteins follow the non- canonical pathway involving tyrosine kinase like orphan receptor (ROR) proteins.
 These proteins have been found to play an important role in stimulating and regulating the role of DFSCs in
forming non-mineralizing PDL and mineralized alveolar bone and cementum

34. Schematic diagram of procedure used to generate engineered
dental root analogue.
The third molar tooth is harvested from the mandible of a 6-
month-old pig to obtain dental follicle, dental pulp, and enamel
organ, and each cell is independently isolated.
DFSCs are subcultured only until sufficient cell numbers for
periodontal tissue regeneration are obtained.
The cylindrical bone cavity is made from pig mandibular bone
shaft.
Firstly, subcultured DFSCs are seeded at
the bottom of the bone cavity.
Then, dental pulp cells, enamel organ epithelial cells, and
subcultured DFSCs are successively placed over the preceding
layer.
A mimic of the tooth germ is thus created with dental cell
populations.
Dental follicle stem cells and tissue engineering
Masaki J. Honda
Journal of Oral Science, Vol. 52, No. 4, 541-552, 2010

35. STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH (SHED):
 In a study by Miura et al in 2003 found that multipotent stem cells
are found in exfoliated human deciduous teeth. Hence, these cells can be
readily harvested. They have a higher proliferation rate as compared to
BMMSC and PDLSC, also result in increased bone formation . SHED
instead of directly forming the specific cells, create a special template
for recruiting host cells, resulting in inducing the new tissue formation
.
35

36.  SHED have been found to elevate regulatory T cells and downregulate T-helper 17 cells, hence have
significant immunomodulatory capacity
 In vitro characterization reveals early MSC markers STRO-1 and CD146.
 Studies by Kerkis, et al. revealed that they are basically immature DPSC and contain cell markers like
Oct-4, Nanog which are present in embryonic stem cell.
 Fu, et al. in 2014 investigated the role of allogeneic SHED in the swine periodontitis model, and found
that they resulted in predictable periodontal regeneration similar to PDLSCs.
 Also, Yamada, et al in 2011 used SHED obtained from puppies and placed them in mandibular osseous
defects created in parent canines. At 8 weeks the defect was completely filled with mature bone. Hence
SHED derived from a child can be successfully used as graft in the parent.
36

37. The obvious advantages of SHEDs are:
a) Higher proliferation rate compared with stem cells from permanent teeth; because they are less mature than
other stem cells found in the body.
b) Easy to be expanded in-vitro.
c) High plasticity since they can differentiate into neurons, adipocytes, osteoblasts and odontoblasts.
d) Readily accessible in young patients.
e) Especially suitable for young patients with mixed dentition.
f) The process does not require a patient to sacrifice a tooth to source the stem cells.
g) There is little or no trauma.
h) stem cells from human exfoliated deciduous teeth are amenable to cryopreservation, meaning that these
cells could be stored for long periods of time on liquid nitrogen
37

38. GINGIVAL MESENCHYMAL STEM CELLS (GMSC)
 Oral MSCs derived from human gingiva (GMSCs) also have been considered as a
promising alternative cell source for periodontal regeneration
 In addition to physical characteristics of gingival fibroblasts they exhibit adherence to
plastic and multi-lineage differentiation potential
 In vitro characterization reveals cell surface markers CD44, CD73, CD90 and
CD105 also stem cell markers such as SSEA-4, STRO-1, CD146, CD166, CD271
and vimentin which is a mesenchymal marker
 In a study done by Yu et al in 2013 demonstrared canine model with class III
furcation defects, the transplanted GMSCs significantly enhanced the regeneration
of the damaged periodontal tissue, including the alveolar bone, cementum, and
functional periodontal ligament
38

39. EPITHELIAL CELL RESTS OF MALASSEZ (ECRM):
 These are remnants of the Hertwig’s epithelial root sheath from which arise,
all the periodontal structures.
 Since ECRM is normally present within the periodontium, hence studies have
been carried out to research the stemness of ECRM
 They express MSC markers CD29, CD44, HSP-90β also embryonic stem cell
markers Oct-4, Nanog and SSEA-4.
 Also Xiong J, et al. has reported their capacity to carry out epithelial
mesenchymal interactions.Following which numerous studies by the same
authors have revealed that ECRM can differentiate into bone, cementum and
periodontal ligament.
39

40. INDUCED PLURIPOTENT STEM CELLS
 Apart from the MSCs, experiments have been carried out to test whether somatic cells can be induced to
transform into pluripotent stem cells.
 Takahashi, et al. showed that adult somatic cells can be transformed into pluripotent stem cells by
forcing expression of certain transcription factors
 Hence, cells obtained from oral epithelium or other dental sources can be easily transformed to the desired
cell type using the required transcription factors. These stem cells have drawn considerable attention
since they are very similar to embryonic stem cells, hence considerable potential in periodontal
regeneration
 Duan, et al. has used these stem cells along with Emdogain in periodontal fenestration defect models
resulting in the complete regeneration of the periodontium.
40

41. Induced Pluripotent stem cells (iPS cells)
cell from the body
‘genetic reprogramming’
= add certain genes to the cell
induced pluripotent stem (iPS) cell
behaves like an embryonic stem cell
Advantage: no need for embryos! all possible types of
specialized cells
culture iPS cells in the lab
differentiation

42. Induced Pluripotent stem cells (iPS cells)
cell from the body (skin)
genetic reprogramming
Pluripotent stem cell
(iPS)
differentiation

43. CELL SHEET ENGINEERING
 Although stem cells may be a promising source of periodontal regeneration, for better results they need to be
transferred to the target site with appropriate scaffold and the correct signaling molecules for them to differentiate
into the desired tissues.
 Conventionally, tissue engineering involved the incorporation of cells, growth factors and scaffold separately into
the defect
 Over the years, researchers attempted at culturing cells in vitro under ideal conditions resulting in formation of cell
sheets which were separated from the substratum by enzymatic treatment, and placed at the target site.
 This however was found to impair cell functions, since the proteolytic enzymes used hydrolyze various membrane
associated proteins resulting in damage to the cell membrane.
43

44. 44
 Pioneering work carried out by Okano et al, in the field of cell sheet engineering helped in
overcoming this issue and making it a viable mode of periodontal regeneration.
 Okano, et al. incorporated a temperature responsive polymer poly(N-isopropylacrylamide)-
(PIPAAm) in the culture dishes to detach the cell sheets.
 Since this polymer is hydrophilic at temperatures greater than 32°C and hydrophobic when
temperature is reduced below 32°C, also cells adhere to hydrophobic surfaces, therefore it is a useful
aid in detaching the cells from the culture dishes.

45. 45

46.  To increase the strength and number of cells, a 3D culture model of multilayered cell sheet have so been developed
 Also more recently cell sheet fragments and cell sheet pellets have been developed to increase the efficacy of the
cells transplanted especially in cases where the target site is too small for the entire cell sheet
 Further Co-culturing and micro-patterning of different types of cells are under trials for creation of more tissue-
like materials which would give better results than single cell-sheets
 Also several researchers have attempted to incorporate biocompatible scaffolds like hyaluronic acid, fibrin gel
and ceramic bovine bone to the fragile cell sheets also referred to as scaffold based cell sheet technology to
improve the results following cell sheet engineering
46

47. Hence cell sheets of the following types have been manufactured till date-
1. Monolayered Cell Sheets (MCS)
2. Multi-Layered cell-Sheet (MLS)
3. Cell Sheet Fragments (CSF)
4. Cell Sheet Pellets (CSP)
5. Co-culturing and micropatterning
6. Scaffold based CST
47

48. DRAWBACKS OF CELL SHEET ENGINEERING
 There are certain drawbacks associated with cell sheet engineering, which need to be addressed prior
to clinical applications in patients.
 These cell sheets are extremely delicate which makes their handling and transportation without
damage extremely difficult.
 Also numerous applications of multilayered cell sheets would be required to resemble the lost or
damaged natural tissue.
 The high cost of this procedure is also a major impediment in making this treatment modality a reality.
48

49. Dental stem cell markers
Stem cell markers are genes and their protein products used by scientists to isolate and identify stem
cells. Stem cells can also be identified by functional assays

50. Application
of
dental stem cells
Craniofacial
regeneration
Cleft lip and
palate
Tooth
regeneration
Pulp
regeneration
Enamel and
dentin
production
Periodontal
ligament
regeneration

51. STEM CELLAPPLICATION IN PERIODONTICS
 The periodontium is an unusually complex tissue comprised of two hard and two soft connective tissues.
 Once damaged these tissues have limited capacity to regenerate.
 The complex series of events associated with periodontal regeneration involves recruitment of locally derived
progenitor cells to the site which can subsequently differentiate into periodontal ligament forming cells,
cementum forming cementoblasts and bone forming osteoblasts .
 To date restoration of damaged and diseased periodontium has relied almost entirely on the use of implantation of
structural substances often with little reparative potential.
 More recently biological approaches based on principles of tissue engineering have emerged as prospective
alternatives to conventional approaches.
51

52. 52
 Such approaches include gene therapy, stem cell therapy, and local administration of biocompatible
scaffolds with or without the presence of selected growth factors.
 The exploration of tooth tissue engineering mainly focuses on three parts, seeding cells, scaffolds and growth
factors.

53. 53
One of the critical requirements of the tissue engineering approach is the delivery of ex vivo expanded progenitor
populations or the mobilization of endogenous progenitor cells capable of proliferating and differentiating into the
required tissues.
 By definition stem cells fulfill these requirements and the recent identification within the periodontal ligament
represents a significant development in the progress towards predictable periodontal regeneration .
 In the field of tooth engineering efforts have been made to explore MSCs, such as stem cells from human exfoliated
deciduous teeth (SHEDs; adult dental pulp stem cells (DPSCs), stem cells from the apical part of the papilla (SCAPs),
stem cells from the dental follicle (DFSCs), periodontal ligament stem cells (PDLSCs), bone marrow-derived
mesenchymal stem cells (BMSCs) and epithelium-originated dental stem cells

54. STEM CELLS IN PERIODONTAL REGENERATION
 Periodontal regeneration can be defined as the complete restoration of lost tissues to the original architecture and
function by recapitulating the crucial wound healing events associated with their development .
 Such processes support the concept that some mesenchymal cells remain in the periodontal ligament and are
responsible for tissue homeostasis, serving as a reservoir of renewable progenitor cells throughout adult life.
 As seeding to enhance regeneration of other tissues seems to be successful, it seems logical that PLSCs cultured
within a suitable delivery scaffold, in conjunction with growth and differentiation factors present in autologous
blood clots, will lead to new periodontal tissue attachment via a tissue engineering approach.
 Studies to date have shown that stem cells can be isolated and characterized within the periodontal ligament.
54

55.  In 2005 Saito et al. achieved success in development of a cementoblast progenitor cell line. Periodontal ligament
cells were transplanted into nude mice on a hydroxyapatite tricalcium phosphate scaffold. Histopathological assays
indicated bone-like tissue formed containing cementocyte-like cells in a mineralized matrix. Thus it is worth noting
that PLSCs may serve as a source of cells for cementum formation.
 Nagatomo et al. in their study showed that PLSCs differentiated into osteoblasts in a dose-dependent manner,
clarifying that tissue stem cells or progenitor cells of osteoblasts are present in cells which make up the periodontal
ligament.
 Orciani et al. verified the osteogenic ability of PDLSCs and pointed out that differentiating cells were also
characterized by an increase of Ca2 + and nitric oxide production. The authors demonstrated that local
reimplantation of expanded cells in conjugation with a nitric oxide donor could represent a promising method for
treatment of periodontal defects.
55

56. 56
 An interesting study by Yamada et al. showed a novel approach to periodontal tissue regeneration with MSCs and platelet rich
plasma (PRP) using tissue engineering principles. The MSCs were isolated from iliac crest marrow aspirates from patients and PRP
was isolated from peripheral blood. A MSC-PRP gel was prepared and applied to root surfaces and adjacent defect spaces.
Reexamination after 1 year demonstrated that application of MSC-PRP at sites with angular defects resulted in reduction of probing
pocket depth by 4 mm, increased in clinical attachment level, while bleeding and tooth mobility disappeared. Radiographic
assessment showed that the bone defects had reduced in depth. Interdental papillas supported by tissue engineering principles were
regenerated.Thus MSC-PRP helped in periodontal tissue regeneration which is aesthetically acceptable and reduced patient
morbidity.

57. AMNIOTIC MEMBRANE
 The amniotic membrane (AM) encases the amniotic fluid and fetus, and is highly flexible because of which it is
easily be separated from the chorion.
 AM has two types of cells with different embryological origins: amnion epithelial cells derived from
embryonic ectoderm and amnion mesenchymal cells from embryonic mesoderm.
57

58. 58

59. PROPERTIES OF AMNIOTIC MEMBRANE
 Anti-inflammatory
 Angiogenesis
 Immunomodulatory
 Anti-microbial and Anti-viral
 Anti-Scarring
 Promotion of epithelialization
 Reduction of pain
 Increased extracellular matrix deposition
59
Mesenchymal
stem cells
 TIMPS –
123 and IL - 10
and IL – 1 RA
 IL- 4 and IL-
10
 TNF – alpha
and IFN

60. It not only maintains the structural and anatomical configuration but also contributes to the enhancement of healing through
reduction of post operative scarring and subsequent loss of function and providing a rich source of stem cells.
Amnion has shown an ability to form a n early physiologic “seal” with the host tissue precluding bacterial contamination
Amnion tissue contains growth factors that may aid in the formation of granulation tissue by stimulating fibroblast growth
and neovascularization
It has an ability to decrease the host immunologic response via mechanisms such as localized suppression of
polymorphonuclear cell migration
LAMININ-5 being the most prevalent plays a role in the cellular adhesion of gingival cells and concentrations of this
glycoprotein is useful for periodontal grafting procedures.
Advantages of using Amniotic membrane over other membrane

61. AMNIOTIC MEMBRANE PLACEMENT

62.  Shetty et al in 2014 compared usage of Platelet-rich Fibrin (PRF) and amniotic membrane in
bilaterally occurring multiple Miller Class I recession. 100% root coverage was observed
with both of the membranes but the results were stable even after seven months in the
amniotic membrane-treated site.
 Samandari et al in 2004 suggested that the amniotic membrane graft might be used as a
potential graft material for vestibuloplasty.
 Kothari et al in 2012 also concluded that grafts of amniotic membrane are viable and reliable
for covering of the raw surface, prevent secondary contraction after vestibuloplasty, and
maintain the postoperative vestibular depth.
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63. LIMITATIONS
1. The use of amniotic membranes requires skill; thus, operator’s inexperience is one
limitation.
2.There is always an associated risk of infection transmission with transplantation of
amniotic membranes. Adequate precautions should be taken and safety criteria should be
included in application of these biological membranes .
3. Amniotic membranes are fragile membranes, so they need to be dealt with very carefully.
Cryopreserved/ hyperdry membranes are expensive .
4.The procedure associated with use of these membranes is technique-sensitive and also
depends on defect morphology.
63

64. 64
Challenges encountered
Biological
• Molecular pathways
Technical
• Culture mediums
• Xenogenic – source of
pathogen
• MSC’s – Limited Life
span
• Ideal biocompatible
scaffold and transport
mechanism
Clinical
• Integration of HSC
derivatives with
recipient tissue - ability
– desired function -
speculation

65. FUTURE DIRECTIONS
 Adequate numbers of stem cells in the target site have
continued to be an impediment to the application of stem
cells for periodontal regeneration.
 Also, newer regeneration strategies such as scaffold based
tissue engineering, modified cell pellet cultures,
bioengineered tooth parts assembly and stem cell based
gene therapy is being examined .
65

66. 66

67. 67

68. CELL SHEET FRAGMENT AND CELL SHEET PELLET
Zhao et al. in 2012 applied CSF of periodontal ligament stem cells (PDLSCs) and platelet-rich fibrin (PRF)
granules in combination for avulsed tooth reimplantation, and in vitro outcomes showed better regeneration of
PDL like tissues and a reduction of ankylosis and inflammation in the PDLSCs/PRF group compared with the
other testing groups, which suggested that the PDLSCs/PRF construct possesses good effectiveness and
clinical potential.
Na et al in 2013 developed a 3D cell pellet cultivation system and manufactured CSPs using stem cells from
root apical papilla, the in vitro test results showed a higher proportion of ECM components and a higher
expression of alkaline phosphatase (ALP), dentin sialoprotein (DSPP), bone sialoprotein (BSP) and runt-related
gene 2 (RUNX2) mRNA than in the cell sheets.
68

69. CO-CULTURE AND MICRO PATTERNING
69
 Bai et al. in 2011 reported successful co-culture of two types of cell sheets in a stratified manner in a
specifically designed chamber, and the results showed that dental follicle stem cells co-cultured with
Hertwig's epithelial root sheath cells had prior effectiveness in periodontal regeneration compared with
cells cultured alone.
 cell micropatterning is also a key method to study cell behavior, cell–cell and cell–ECM interactions.
 Until now, there have been several methods of micropatterning, including
photolithography,microfluidics, microfabrication of stencils and microcontact Printing.
Micropatterning is a promising approach to the creation of a pre-designed microenvironment for cells and
ECMs and for the spatio-temporal control of cell-to-cell interactions in vitro,

70. SCAFFOLD-BASED CST
 While considering the fragile structure of cell sheets, investigators deem that scaffolds may
be necessary for supporting them in implantationor 3D-tissue reconstruction.
 Scaffolds have great influence on the fates of seeding, such as cell adhesion, proliferation,
migration, differentiation and function
 Vaquette et al. in 2012 designed a biphasic scaffold composed of a Fused Deposition
Modeling scaffold (bone compartment) and an electrospun membrane (periodontal
compartment) for periodontal regeneration to achieve simultaneous alveolar bone and
periodontal ligament regeneration.
70

71.  Careful investigations need to be carried out to guarantee that the MSC would not form neoplasms
 To be able to achieve the extracellular matrix in the target site should generate the correct signals
at the appropriate time for all the tissues to form.
 However, further investigations need to be carried out to assess the feasibility of the use of
allogeneic MSC considering that in the clinical environment, complete regeneration needs to occur
in a diseased environment containing inflammatory cytokines.
 Further on various interactions between the different types of cells in the periodontium need to be
assessed and also the effect of mechanical stress on periodontal regeneration needs to be examined
in detail.
71

72. 72
CONCLUSION
As the research in this field continuing
on with promising future - we may be able
– in the near future – to implant a bio
tooth primordium into a patient’s gum in
the place of a removed or lost tooth

73. 73
 We are just at the beginning of a very long road of work and discovery, but
one thing is certain - the research on stem cells – the precursors for life is vital
and must go on.
 Hence to conclude:
“Pro Life Paves Path For Life”

74. REFERENCE
 The Scope of Stem Cells in Periodontal Regeneration Thomas George V*, Nebu George Thomas, Saumya John and
Prameetha George Ittycheria - June 05, 2015
 Stem cells in periodontics A futuristic reality Sheetal Oswal, S. Ravindra, Sahitya Sanivarapu J. Stomat. Occ. Med.
(2011) 4:95–104.
 CAVENDER, A. C., APOS, ASOUZA, R. N., GALLER, K. M., KOEKLUE, U., SCHMALZ, G. & SUGGS, L. J.
2011. Bioengineering of dental stem cells in a PEGylated fibrin gel. Regenerative Medicine.
 LYMPERI, S., LIGOUDISTIANOU, C., TARASLIA, V., KONTAKIOTIS, E. & ANASTASIADOU, E. 2013. Dental
Stem Cells and their Applications in Dental Tissue Engineering. The open dentistry journal.
74

75.  ROSA, V., DELLA BONA, A., CAVALCANTI, B. N. & NÖR, J. E. 2012. Tissue engineering: from research to dental
clinics. Dental Materials, 28, 341-348.
 MORAD, G., KHEIRI, L. & KHOJASTEH, A. 2013. Dental pulp stem cells for in vivo bone regeneration: A
systematic review of literature. Archives of Oral Biology, 58, 1818-1827
 Barbara, Zavan, et al. "Dental Pulp Stem Cells and Tissue Engineering Strategies for Clinical Application on
Odontoiatric Field." (2011).
 Malhotra, Neeraj, and Kundabala Mala. "Regenerative endodontics as a tissue engineering approach: Past, current
and future." Australian Endodontic Journal38.3 (2012): 137-148.
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76. Thank YOU
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