This document provides an overview of tissue engineering as it relates to periodontal regeneration. It discusses the historical perspective of tissue engineering and need for strategies to regenerate lost periodontal tissues. Current strategies employed include using scaffolds, growth factors, and stem cells. Scaffolds provide a framework for cellular migration and integration. Sources of cells for tissue engineering include mesenchymal stem cells and various dental stem cells. Growth factors that have potential for periodontal regeneration include PDGF, IGF, TGF-β, and FGF. Future research directions include whole tooth regeneration using dental progenitor cells and scaffolds.

1. Dr. Thaslim Fathima
2nd YEAR POST-GRADUATE

2. CONTENTS
• INTRODUCTION
• HISTORICAL PERSPECTIVE
• NEED FOR TISSUE ENGINEERING
• CURRENTLY AVAILABLE STRATEGIES
EMPLOYED TO ENGINEER TISSUE
• SCAFFOLD OR SUPPORTING MATRICES
• SOURCES OF CELLS IN TISSUE ENGINEERING
• SIGNALING MOLECULES IN TISSUE
ENGINEERING
• FUTURE DIRECTIONS/ CONSIDERATIONS
• CONCLUSION
• REFERENCES

3. INTRODUCTION
Regeneration of the periodontal tissues
is a complex phenomenon requiring
interplay between various processes in
a timely manner.
Langer and collegues 1993
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Tissue engineering is an interdisciplinary field that
applies principles and methods of engineering and the
life sciences towards the development of biological
substitutes that restore, maintain, and improve the
function of damaged tissues and organs.
Term ―tissue engineering was coined at a
National Science Foundation (N.S.F.)
bioengineering meeting in Washington D.C

4. Periodontal regeneration
In the past few decades, many attempts have been made to unravel the
“magic filler” material that could result in new clinical and histological
attachment, but have only culminated in healing by repair.
Periodontal repair refers to healing that does not allow the original
morphological nor functional restoration of the tissue, considered as
non-functional scarring.
Tissue engineering was proposed as a possible technique for
regenerating lost periodontal tissues by Langer and colleagues in 1993.
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5. DEFINITION
Defined as the reconstruction of living tissues, to be used for replacement
of damaged or lost tissue/organs of living organisms; and is founded on
the principles of cell biology, development biology and biomaterial
sciences.
(Nerem R, Sambanis A, 1995)

6. DEFINITION
As the application of principles and methods of engineering and life sciences
towards the fundamental understanding of structure-function relationships, in
normal and pathological mammalian tissues and the development of biological
substitutes that restore, maintain, or improve tissue function.
(National Science Foundation workshop)

7. Historical perspective
• An important component in the early development of tissue engineering was the parallel
development of artificial biomaterials.
• In the mid-1960s, artificial skin and synthetic fibers were being tried as artificial skin grafts for burn
treatment.
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8. Historical perspective
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• In the early 1970s, there were concerted efforts to treat artificial surfaces to be used in implants in
ways that would enable them to avoid causing blood coagulation, by applying special heparin
complex coatings.
• One year later, in 1985, Y.C. Fung proposed that the NSF establish a new research center to be known
as the “Center for the Engineering of Living Tissues”.
• In 1988, Joseph Vacanti and Robert Langer published what would be the first of many tissue
engineering papers together — a study of cell transplantation using bioresorbable scaffold carriers.

9. GOAL OF TISSUE ENGINEERING
Availability
of cell types
needed
Presence or
absence of
cues or
signals
necessary
to recruit
and
stimulate
these cells
The goal of tissue engineering is to
promote healing, and ideally, true
regeneration of a tissue's structure and
function, more predictably, more
quickly, less invasively, and more
qualitatively than allowed by previous
passive technique.

10. STRATEGIES TO TISSUE
ENGINEER
Conductive approaches utilize biomaterials in a passive manner to
facilitate the growth or regenerative capacity of existing tissue.
Example- use of barrier membranes in guided tissue regeneration.
Barrier membrane exclude connective tissue cells that will interfere
with the regenerative process, while enabling the desired host cells
to populate the regeneration site
-Conductive -Inductive -Cell transplantation approaches.

11. Osteoinduction implies the recruitment of immature cells and
the stimulation of these cells to develop into preosteoblasts.
Uses a biodegradable polymer scaffold as a vehicle to deliver
growth factors and genes to the host site.
Chemical process by which molecules contained in graft
convert the neighbouring cells into osteoblasts which inturn
form bone. Eg BMP

12. • Involves direct transplantation of cells grown in the
laboratory.
• It truly reflects the multidisciplinary nature of tissue
engineering, as it requires the clinician or surgeon, the
bioengineer, and the cell biologist.
• Cell transplantation strategy uses a vehicle for delivery in
order to transplant cells and partial tissues to the host
site.

13. TISSUE ENGINEERING TRIAD

14. Giorgio Iviglia
J. Funct. Biomater. 2019

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o(I) The stimulation of the growth of the key surrounding tissues by
applying barrier membranes and bone grafting materials and
o(ii) The prevention of the growth and proliferation of undesired cell types
like epithelial cells.

17. SCAFFOLDS
Scaffold
Framework to support cellular
migration into the defect from
surrounding tissues.
Delivery vehicle for
exogenous cells, growth
factors, and genes.
May structurally reinforce the
defect to maintain the shape
of the defect.
Serves as a barrier to prevent
infiltration of surrounding
tissue that may impede the process
of regeneration.
Before its absorption, a
scaffold can serve as a matrix
for exogenous and
endogenous cell adhesion
Facilitates and regulates
certain cellular processes,
including mitosis, synthesis
and migration

18. Ideal Characteristics of scaffolds
Scaffold
Easy cell
penetration,
distribution and
proliferation
Permeability of
the culture
medium
Maintenance of
osteoblastic cell
phenotype
In vivo
vascularization
( once implanted)
Adequate
mechanical
stiffness
Proper
biodegradation
Ease of
fabrication

19. BIOMATERIALS USED AS SCAFFOLDS
Scaffold
Absorbable
Synthetic
polymers
Natural
polymers
Natural
mineral
Non
resorbable
Synthetic
polymer
Synthetic
ceramics

20. BIOMATERIALS USED AS SCAFFOLDS

21. Currently, computer-assisted design/computer-assisted manufacturing
(CAD/CAM) and rapid prototyping techniques allow the generation of custom-
made scaffolds for cell delivery that fit into certain bone defects.

22. • Liao et al. in a study compared porous beta-tricalcium phosphate/chitosan composite scaffolds
with pure chitosan scaffolds.
Composite scaffolds showed higher proliferation rate of human periodontal ligament cells
(HPLCs) and up-regulated the gene expression of bone sialoprotein and cementum attachment
protein.
In vivo, HPLCs in the composite scaffold not only proliferated, but also recruited vascular tissue
ingrowth; thus, suggesting the benefit of using these composite scaffolds
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23. Jin et al (2004) in an experimental study on dogs has demonstratred that basic fibroblast growth factor with
a controlled release system developed according to the new concept of "in situ tissue engineering."
containing sandwich membrane composed of a collagen sponge scaffold and gelatin microspheres was
able to regenerate periodontal tissues.
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24. CELLS
• Cell source is an important parameter to consider when applying tissue
engineering strategies to restore lost tissues and functions.
CELL
SEEDING
CELL
SUSPENSION

25. • Cell incorporation into implantable matrices, which ensures their localization at
treatment site – concept being referred to as cell seeding.
• An alternative is to inject a cell suspension into sealed compartment containing
defect.

26. SOURCES
Sources
Autologous
cells
Allogenic
cells
Xenogenic
cells
Stem cells

27. Stem cells
Stem cells are immature progenitor cells capable of self
renewal and multi-lineage differentiation through a
process of asymmetric mitosis that leads to two daughter
cells, one identical to the stem cell (daughter stem cell)
and one capable of differentiation into more mature cells
(progenitor cells).

28. CLASSIFICATION (BASED ON DIFFERENTIATION)
Multipotent: the potential to give rise to cells from multiple, but a limited number of lineages.
E.g.: mesenchymal stem cells
Oligopotent: the capacity to differentiate into a few cell types.
E.g. Myeloid stem cells
Unipotent: the ability to differentiate into only one type of cells.
E.g. Skin.

29. ADULT STEM CELLS EMBRYONIC STEM
CELLS
CLASSIFICATION
(BASED ON THE SOURCE OF ORIGIN)
Depending on the development stage of the tissues from
which the stem cells are isolated

30. FRIEDENSTEIN AND COLLEGUES
o Embryonic stem cells are derived from
embryos that are 2 – 11 days old called
blastocysts. They are totipotent cells….
o Adult stem cells are multipotent stem
cells, and depending upon their origin,
they can be further classified into
hemopoetic stem cells and mesenchymal
stem cells.
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31. MESENCHYMAL STEM CELLS
Hold great
potential for
autologous cell
based therapy
Capable of multi
lineage
differentiation
Adherent,
proliferating
Differentiates into multiple tissue types, including bone, cartilage, muscle, tendon.

32. • Another important characteristic of MSCs for regenerative medicine is their potential
allogenic use without immunosuppressive therapy.
• Within the sphere of periodontal tissue engineering, mesenchymal derived cells have
been applied for simultaneous regeneration of the attachment apparatus components.

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DENTAL STEM CELLS

34. DENTAL PULP STEM CELLS
• Human pulp cells (odontoblasts) retain its
ability to form functional odontoblast
even when after fully developed complete
tooth development.
• It has the ability to form reparative dentin
when expose to deep caries and mild
trauma or pulp capping.
2003, Shi and Gronthos

35. • Easy surgical access to the collection
site and very low morbidity after
extraction of the dental pulp.
DPSCs can generate much more typical
dentin tissues within a short period than
nondental stem cells.
Can be safely cryopreserved and
recombined with many scaffolds.
Possess immuno-privilege and anti-
inflammatory abilities favorable for the
allotransplantation experiments.
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36. Stem cells from human exfoliated deciduous teeth
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• Dr. Songtao Shi discovered SHED in 2003.
• Miura et al. confirmed that SHED were able to differentiate into a variety of cell types to a
greater extent than DPSCs, including osteoblast-like, odontoblast-like cells, adipocytes, and
neural cells.
• Abbas et al.investigated the possible neural crest origin of SHED.
• The main task of these cells seems to be the formation of mineralized tissue, which can be
used to enhance orofacial bone regeneration

37. Stem cells from apical papilla (SCAP)
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• MSCs residing in the apical papilla of permanent teeth with immature roots are known
as SCAP.
• These were discovered by Sonoyama et al.
• SCAP are capable of forming odontoblast-like cells, producing dentin in vivo, and are
likely cell source of primary odontoblasts for the formation of root dentin.
• SCAP supports apexogenesis, which can occur in infected immature permanent teeth
with periradicular periodontitis or abscess.
• Complete root formation is seen under the influence of the surviving epithelial root
sheath of Hertwig.

38. PERIODONTAL LIGAMENT STEM CELLS
• Multipotent progenitors from human PDL were shown to generate bone.
• Exhibit the ability to differentiate into cementoblast- like cells, adipocytes and
fibroblasts.
• These cells have also been shown to retain stem cell properties and tissue
regeneration capacity even after recovery from solid- frozen human primary
tissue (Shi 2005).
• The principle of guided tissue regeneration is based on this principle that
periodontal ligament cell have the potential to give rise to various cells.
• Liu et al, 2008 Autologous periodontal ligament derived mesenchymal stromal
cells promoted healing of experimental periodontitis in mini-pigs.
• These findings suggest that cryopreserved PDLSCs from extracted teeth could
prove useful for clinically relevant therapeutic applications in the future.

39. WHOLE-TOOTH REGENERATION
• Whole-tooth regeneration efforts largely consist of two approaches:
• One involves in vivo implantation of immature tooth structures grown in vitro from dental
progenitor cells,
• While the other uses in vitro expanded, cultured dental progenitor cell populations seeded
onto polymer scaffolds and implanted in vivo.

40. General schematic representation of the current strategy for whole tooth
regeneration using iPS cells

41. TOOTH STEM CELL BANKING
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• Although tooth banking is currently not very popular the trend is gaining acceptance
mainly in the developed countries.
• The first tooth bank was established in Hiroshima University and the company was
named as “Three Brackets” (Suri Buraketto) in 2005.
• Stemade introduced the concept of dental stem cells banking in India recently by
launching its operations in Mumbai and Delhi.

42. Signaling molecules
• The two types of signaling molecules that have received the greatest attention are
growth factors and morphogens that act by altering the cell phenotype.
• These have pleotropic effects some of which include:
• Mitogenic
• Chemotactic
• Angiogenic effects

43. Growth factors

44. Platelet-derived growth factor
• It was discovered by Lynch and coworkers in the late 1980s.
• Platelet-derived growth factor (PDGF) is the natural wound healing “hormone”.
• PDGF secreted from platelets play an important role in initial wound healing, its
subsequent secretion from macrophages continues the events of wound healing
through up-regulation of other growth factors and cells that ultimately promote
fibroblastic and osteoblastic functions.

45. • Moon et al. applied PDGF-BB to promote migration and proliferation of periodontal
ligament fibroblasts.
• They demonstrated that PDGF has the capacity to stimulate bone formation and
periodontal regeneration in vivo and indicate that it holds promise as an important
adjuvant to periodontal surgery.

46. Insulin like growth factor
• Insulin like growth factor-I is found in substantial levels in platelets and is
released during clotting along with the other growth factors.
• Han and Amar demonstrated that in vitro IGF-I substantially enhanced cell survival in
periodontal ligament fibroblast compared to gingival fibroblasts by the up-regulation of
anti-apoptic molecules and down-regulation of pro-apoptotic molecules.

47. Transforming growth factor
• TGF-β appears to be a major regulator of cell replication and differentiation.
• Three forms of TGF-β have been identified namely TGF-β1, TGF-β2, and
TGF-β3.
• It is chemotactic for fibroblasts and cementoblasts, and promotes fibroblast
accumulation and fibrosis in the healing process
• TGF-β isoforms have multiple regulatory roles in the synthesis, maintenance
and turnover of the extracellular matrix.

48. Oates et al. compared the
mitogenic activity of TGF-β with
interleukin-1 and PDGF in
fibroblast cells derived from
periodontal ligament explants.
TGF-β was relatively a weak mitogen for
Periodontal (PDL) cells compared to PDGF,
suggesting that TGF-β may indirectly
stimulate DNA synthesis

49. Fibroblast growth factor
• The two most thoroughly characterized forms are: Basic FGF (bFGF) and
acidic FGF (aFGF).
• Both aFGF and bFGF are single chain proteins that are proteolytically
derived from different precursor molecules to generate biologically active
proteins of 15,000 molecular weight.
• They promote proliferation and attachment of endothelial cells and PDL
cells in wound healing process.
• FGF-2 is known to attract epithelial cells more effectively than FGF-1

50. Examined the efficiency of topical
application of FGF-2 with
periodontal regeneration in the
bony defects by surgically creating
furcation class II bone defects in
non-human primates and
concluded that a topical
application of FGF-2 can enhance
considerable periodontal
regeneration.
Takayama et
al.
Did a recent randomized clinical trial
trying to evaluate the therapeutic
response to varying doses of FGF-2
(bFGF).
They demonstrated a significant
increase in the alveolar bone height
on using 0.3% FGF-2.
Kitamura et
al.
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51. Hepatocyte growth factor
• Hepatocyte growth factor (HGF) is a
secreted, heparin sulfate
glycosaminoglycan-binding protein.
Yamada et al. cultured fibroblasts in a culture medium
containing HGF and concluded that they produced good cell
proliferation and vascular endothelial growth factor (VEGF)
release.
The results suggest that it may provide a new tool for the
treatment of gingival recession

52. Bone morphogenetic proteins
• They trigger cellular effects by way of heterotetrameric serine/ theonine
kinase receptors and intracellular signaling proteins.
• They are a group of related proteins that are known to possess the unique
ability to induce cartilage and bone formation.
• BMPs, like PDGF, play a role in the blood vessel formation.

53. • They play an important role in the angiogenetic activity by up-regulating the
angiogenetic peptides like VEGF, may bind to endothelial cells and stimulate
the migration and promote blood vessel formation.
• The hallmark property of BMP is the differentiation factor.

54. CLINICAL APPLICATIONS
OF TISSUE ENGINEERING
FOR PERIODONTAL TISSUE
REGENERATION

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56. GUIDED TISSUE REGENERATION
• Nyman and Karring in the 1982 were the first ones to have proposed the use of guided tissue
regeneration for periodontal regeneration, which marked the evolution of periodontal
regeneration technologies using tissue engineering.
• The placement of barrier membranes over the denuded root surface and the debrided
periodontal defect has shown space provision, epithelial cell occlusion, exclusion of gingival
connective tissue from the root surface and selective repopulation of periodontal ligament
cells.

58. Enamel matrix derivative
Commercially available as Emdogain which have been known to effect
periodontal regeneration
Recent data from a systematic review indicates that biologically EMPs cause an
increase in cell attachment of epithelial cells, gingival fibroblasts, and PDL
fibroblasts.
Stimulation in the synthesis of total protein and extracellular matrix molecules
has also been documented

59. • The rationale for the clinical use of enamel matrix derivative is the observation that enamel
matrix proteins are deposited onto the surfaces of developing tooth roots before cementum
formation.
They increase the expression of transcription factors that are related to chondroblast and
osteoblasts/cementoblast differentiation
Use of Enamel matrix derivative (EMD) and a demineralized freeze dried bone allograft
(DFDBA) have been demonstrated to be osteopromotive in nature; thus, resulting in an
additional increase in bone formation.
The only concern with the use of EMD has been related to its application and its related
viscous nature, which may not provide sufficient soft tissue/flap support; thus, potentially
limiting the space available for the regeneration process.

61. Platelet rich plasma
• Platelet rich plasma (PRP) is a volume of autologous plasma that contains a
platelet concentration above baseline values.
• The development of PRP from autologous blood by simple, sterile centrifugation
produces a concentration of platelets with enhanced growth factors including
PDGF, TGF-β, and insulin growth factor-1.
• It has been reported that PRP preparations may increase the concentrations of
platelets up to 38%.

62. • PRP works through transmembrane receptors and intra cytoplasmic signaling
pathways, as do all other growth factor preparations.
• PRP stimulates the proliferation of human osteogenic cells and periodontal
ligament cells.
• Because of its fibrinogen content, PRP reacts with thrombin and induces fibrin
clot formation, which, in turn, is capable of upregulating collagen synthesis in
the extracellular matrix and provides a favorable scaffold for cellular migration
and adhesion.

63. Various growth factors in PRP
Platelet-derived Growth
factor
Transforming growth
factor-beta Insulin-like growth factor
Basic fibroblast growth
factor-2 Epidermal Growth factor Vascular endothelial
growth Factor
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Recombinant protein therapeutics
To date, only three recombinant growth factor products have
been widely used
rh PDGF-BB (gel) rhPDGF-BB (with β
tricalcium phosphate)
rh BMP-2 (with type I
collagen sponge)

66. • Recombinant platelet derived growth factor (rh PDGF BB) is more than 98% pure
recombinant protein developed using conventional recombinant expression techniques
under highly controlled conditions.
• Use of rh PDGF has been one of the options to regenerate periodontium and has received
FDA clearance for use.

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o The concept of the use of recombinant protein therapeutics
delivered in an allograft matrix has provided significant clinical
results.
o The efficacy of GEM 21S (growth enhanced matrix β
TCP+PDGF), biomimmetic therapeutics were recently
reported by Nevins and co workers

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Studies have also suggested that the use of rh PDGF+ β
TCP and a collagen membrane may represent an acceptable
alternative to connective tissue graft for covering gingival
recession defects.
o Simion M conducted a study using rh PDGF in conjunction
with anorganic bone block for vertical ridge augmentation.
o It resulted in better healing and increased amount of regenerated
Bone.

71. CELL BASED APPROACHES
• Typical cell harvesting methods using enzymatic dispersion might destroy critical cell surface
proteins such as ion channels, while growth factor receptors and cell to cell junctions remain
intact.
• Cell transplantation using autologous cells is expected to play a central clinical role in the
future. Dental cell seeding attempts have attempted to regenerate the periodontal tissues
since 1990s.
• Okano et al. developed temperature responsive culture dishes by grafting a polymer poly N
isopropylacylamide (PIPAAm) onto tissue culture graded polystyrene dishes by irradiation
with an electron beam.
• Cells generally adhere to hydrophobic surfaces, but not to hydrophillic surfaces. At
temperatures lower than 32°C, it is fully hydrated.

72. • This dish allowed intact cells with preserved extracellular matrix proteins and
normal cell functions to be harvested with just low temperature treatment.
• This has evolved into a novel strategy called “Cell sheet engineering” which
produces tissues without a specific scaffold.
• Transplanted cell sheets can be grafted to the recipient tissues without
suturing.

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Akizuki investigated periodontal healing
after application of PDLcell sheet in
beagle dogs. Results showed that, in
experimental group periodontal tissue
healing with the formation of bone, PDL
and cementum occurred in 3 out of 5
defects.
Flores et al. evaluated whether human PDL
cell sheet could reconstruct periodontal
tissue and found that transplanted PDL cell
sheet cultured with osteogenic differentiation
medium induced periodontal tissue
regeneration containing an obvious
cementum layer and Sharpey’s fiber.
Hasegawa et al. assessed the ability
of periodontal ligament cell sheets to
regenerate the periodontal ligament
tissue and demonstrated its
usefulness in periodontal tissue
Regeneration.
Huang and Zhang have set forward a
hypothesis of transplanting PDL cell obtained
from the periodontium of autogenous
extracted teeth, such as the third molar and
premolar for orthodontic purposes sheets
when cultured using the cell sheet
engineering approach into the implant beds
before inserting the implants.

75. Gene delivery based approaches
• These techniques involve a gene encoding. A therapeutic protein being introduced
into the cells which can then express the target protein.
• This technique avoids the problems associated with the protein delivery method by
maintaining constant protein levels at the site of the defect.

76. CHALLENGES AHEAD
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77. • Structural and functional complexity
of the periodontium.
The fact that more than one tissue
must be reconstructed, namely
alveolar bone, periodontal ligament,
root cementum, and gingiva, makes
it much more difficult to find both the
right combination and the doses of
growth factors.
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78. To overcome the rapid clearance of growth factors, a carrier system must be
found that stores and releases the growth factors over a longer period of time
so that their resident time is prolonged.
Although many carrier systems have been tested, none of them appears to be
ideal.
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79. While high developmental and therapeutic costs appear justified
for severe skeletal conditions such as non-unions, open fractures,
spinal fusion, and large bone defects, for example in the
mandible, the same cannot necessarily be said for relatively small
and non-life-threatening periodontal defects where preventive and
maintenance measures are still mandatory.

80. CONCLUSION

81. References
o Tissue engineering: A new vista in periodontal regeneration Nymphea Pandit, Rajvir Malik,
Deepa Philips,2017.
o Current overview on dental stem cells applications in regenerative dentistry.Ramta Bansal and
Aditya Jain
o Inside Periodontal Regeneration—A More In-Depth OverviewGiorgio Iviglia
J. Funct. Biomater. 2019
o Tissue engineering. Applications in oral and maxillofacial surgery and periodontics- Samuel E
Lynch, 2nd edition
o Schroeder HE. The periodontium. Handbook of microscopic anatomy. Vol V/5. Berlin: Springer,
1986.
o Newman MG, Takei HH, Kloklevold PR, Carranza FA. Carranza’s clinical periodontology. 10 th
ed. WB Saunders Co; 2006.
o Olive G, Chaumanet G, Genovese MD, Beneduce C, Andrena S. A clinical retrospective
evaluation of 156 consecutive cases. G Dent 2010; 58:126-133.
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82. !