This document summarizes key concepts in periodontal regeneration and tissue engineering. It discusses the appropriate cell types and signals needed for periodontal regeneration, including epithelial cells, fibroblasts, and osteoblasts. Scaffolds provide structure for cell attachment, proliferation and differentiation. Growth factors like BMPs, IGFs, FGFs, and TGF-β play important roles in cell differentiation and bone regeneration. Vascularization is also important to provide nutrients and remove waste. Tissue engineering aims to recreate tissues using the principles of cells, signaling molecules, and biomaterial scaffolds.

1. P.Prathahini,
IIIrd MDS
Tissue engineering and
Periodontal Regeneration

2. Introduction
S
e
e t
h
e h
o
l
d
e, fill it ,with anything
-Perio2000vol50.
PrincipleObjective…
“T
ore-create functional, healthy tissues and organs
in orderto replacediseased,dying, ordeadtissues”

4. Periodontal Regeneration
• Appropriate cell types& Signals
• Local environment
– Recruitment of theright cells and
preventing thewrong cell
(Local Environment includes
Cementummatrix & C.E.J).
- Affects cellmigration ,adhesion,
proliferation & differentiation.

5. Epithelial cells –
junctional
epithelium
Fibroblasts –
gingival
&periodontal
ligamentfibres
Blastic cells -
Osteoblasts for
Alveoalr bone,
cementoblasts
for cementum
Cells
Growth factors – FGF-1 &
2, IGF-1&2, BMP, EGF,Pdgf
Adhesion molecules –
fibronectin, osteopontin ,
laminin, bsp, collagens,
CAP
Structural proteins -
Types I,III,V, XII and XIV
collagens, Proteoglycans,
Hyaluran, tenascin, non-
collagenous proteins,
osteonectin,
dentin/enamel proteins
Molecules

6. T
o
sayperiodontalregenerationhasoccurred4criteriashouldb
e
fulfilled
• Functionalepithelialseal
• N
ewconnectivetissu
eattachmen
t
• Acellularextrinsicfiberce
me
n
t
um
• Alveolarboneheightrestored

7. Reasons for Failures in Periodontal
Regeneration technique
• Formation of alongjunctional epithelium;
• Inadequate seal ;
• Wound closure
• Restriction of regeneration.
• Precisedefinition
• Sufficient discrimination
• Infection

8. - Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissue/organs of living organisms and is founded on the principles
of cell biology, developmental biology and biomaterials science
(NaremR. et al.,1995 ;RossoF. et al., 2004;
- “an interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore, maintain, or improve tissue function or a
whole organ"
Langerand Vacanti
T
issu
ee
ngin
e
e
ring

9. • Sixteenth century,of Bologna,
Italy,
“De Custorum Chirurigia per
Insitionem,”
• 1970 – WT Green (Orthopaedic surgeon)
–
created cartilage.
• 1986 – Karl Meyer - created a skin
substitute by using a collagen matrix
• Dr.Langler - designed appropriate

10. Keyco
m
p
o
n
e
n
t
s
Scaffold
Progenitor cells
Biosignals

11. Diagrammaticrepresentationofarteriovenous
shuntloopmodelin t
h
erat– Mian Re
tal 2000
Hole of insertion
of vessel loop
Venous graft
Artery
Vein
Transparent chamber
Femoral vessels Leg

12. Two main criteria for successful tissue
engineering
• Scaffold,
• Architectural geometry
• Space-maintaining
properties
Biomechanical properties Biological functions
• Cell recruitment,
proliferation, survival in
culture and at the site of
implantation,
• Neovascularization
• Delivery of
morphogenetic-,
regulatory- and growth
factors

13. StemCells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types.

14. P
otency
T
h
epotencyspecifiest
h
edifferentiationpotentialoft
h
e
stemcell.
• Totipotent stemcells
• Pluripotent stemcells
• Multipotent stemcells
• Unipotent stemcells

15. T
ypesofStemc
e
lls
Adult
stem
cells….
Embryonic
stem cells

18. invitro
ADVANTAGES
Theability to
examine thematerial
asit is formedand to
performspecific
measurements prior
to implantation.
DISADVANTAGES
- Theabsenceof a
physiologic& mechanical
environment during the
formation of tissuein vitro.
- Union of theimplanted
tissueswith thehost organ
requiresremodeling –
degradationand new tissue
formation at theinterface
of implant with thehost
tissue.

19. invivo
placeunder the influence
of physiologic mechanical
environment.
Incorporation of the
tissues beingformedwith
the surrounding structures.
ADVANTAGES
DISADVANTAGES
Tissue formation takes
Regeneratingtissues may
bedislodgedordegraded
by the mechanical forces
normallyacting at the site,
beforethe regenerating
tissue is fully formedand
incorporated.

20. I)Hem
a
top
oiet
ics
t
e
mc
e
lls
2) Bonemarrow-
stemcells
-Friedenstein et al 1976
-All cell lineages
-Mesenchymal fibroblasts cells
isolated
- Specific surface markers

21. Locally deriveduncommittedcells
• Periodontal ligament stem cells
• Dental pulp stem cells
- Cell surface markers ( Gronthos & co workers 2005)
Thedentalpulp stem cellsrepresentaclonogenicand highly proliferative cell
population
- Denselycalcifiednodules= in vitro.
- Dentinlikeand dentin sialo-phospho-proteinrichmineralisation=
Invivo

22. P
eriodontal ligament ste
mc
ells
• Highly fibrous and vascular…. High turn over
rates
- Cementum like mineralized structure formed
in vitro…
Progenitor cells ….identified in vivo cell
kinetic studies…
• Enriched locations like around the blood
vessels
• Exhibit stem cell features
– Small size
– Responsiveness to stimulating factor
– Slow cycle time.
Most compellingevidence:McCullochand coworkers
1985,1987,1995,1996

23. • Fibroblastic colony forming units …..greater in
PDL stem cell (170) as against the bone marrow
stromal cell (14).
– Propensity …or ….difference in turn over rate.

24. Soe et al, 2004 Isolated a population of multipotent stem
cells in human PDL derived mesenchymal
stromal cells.
Liu et al, 2008
Autologous periodontal ligament derived
mesenchymal stromal cells promoted
healing of experimental periodontitis in
mini-pigs
Studies

25. Growthpotential ofpdl stemcells
• Differential proliferative capacity…
- 80%.... Failed beyond 20 population doublings.
• Pdl Stem cells Versus the Bone marrow stem
cells ..
- 30% higher osteogenic potential ( Yu BH et al 2014)
-BMSCs owned the stronger immunomodulation in
local microenvironment via anti-inflammatory
functions, compared to PDLSCs. (Zhang J et al 2014)
• Finite lifespan of Pdl stem cells…
– Postnatal stem cells (Adult stem cells)
– Embryonic stem cell…virtually immortal
• Genetic manipulation can be done with caution..

26. Characterization andorigin ofPeriodontal
stemcells
• Differentiated from Dental Follicle during embryogenesis….
• Putative cell marker .. STRO-1.. Common
– Isolation using immunomagnetic …fluorescence activated
cell selection.
• Share common expression of cell marker CD146
• McCulloch et al……presence of perivascular progenitor cells in
Pdl

27. Scaffolds/Matrices
• The extracellular matrix consists of
proteins and glycoproteins such as
collagens, laminins, fibronectin, and
proteoglycans and also the
Composition ,
organisation &
distribution of
extracellular matrix
Simple mixing
polysaccharide hyaluronic acid.
Cell isolation
& expansion
Cell on matrix
New tissue

28. Provides...
• Hydration for cells
• Elastic network
• Cell attachment, proliferation &
differentiation
• Store & protect Growth factors from
degradation

29. Interactionsb
e
t
w
e
e
nECM&c
e
ll surafcerecptors
Embryonic
morphogenesis
Cell surface
receptor
Feedback
to ECM
Gene
expression
ECM
Receptor ligand
binding
Receptor GF
binding
Presenting
GF , Cytokines
Storage Pool
Modulation
of ECM
Intracellular
Dynamic
reciprocity
ECM
Cytoskeleton

30. Areac
o
d
ehypothesis...
ECM proteins
• RGD domains in
fibronectin,
vitronectin, YIGSR
domain - laminin,
and GER domain –
type I collagen
Cell surface
receptors
• Integrins,
syndecan ,
CD44,
thrombomoduli
n , laminin
binding protein
, CD36, and
matrikines
• Hood L et al 1977
• The presence of a recognition system that guides cell
positioning
Cell adhesion
MMPs–
b
r
e
a
k
d
o
w
n
and
re
m
o
d
e
llin
go
fECM
...

31. Ide
a
l pro
p
erties
• Biocompatibility (no immunogenicity)
• Space maintenance
• Mechanical rigidity
• Degradability-in a phased manner
• Mechanical structure-three dimensional structure
with sufficient porosity , surface roughness,greater
surface energy,and availability of surface molecules.

32. Natural
• Collagen, hyaluronan, chitosan.
Gelatin, fibrin, alginate, biocover &
Matrix extracts like Matrigel.
• Biodegradable and non-toxic
• Possess known cell binding sites.
• Disadvantages – Immunogenicity ,
speed of degradation and limited
ability to tailor certain specific
properties.

33. S
yn
thetic
• Poly(glycolic acid), poly(lactic acid) or
poly(lacticacid)/poly(glycolic acid) and
their copolymers, poly(p-dioxanone) and
copolymers trimethylene carbonate
• Hydroxyapatite, calcium sulfate, and
tricalcium phosphate - Osteoconductive
• Bioactive glass – silicon network
structure
• Can modify the strength, pore size and
stability.
• Low pH hinder the cell growth
Bioactive glass

34. Collagen sponge scaffold +
gelatin microspheres loaded
with FGF 2 in alveolar bone
defects - Nakahara et al. 2004
Vascularization,
and recovery of
osteogenesis,
periodontal
Collagen sponges + periodontal
ligament cells = cementum had
uniformly regenerated along the
root surface of bony defects in
dog teeth
ligament function after 4 weeks.
Sculean et al. 2003, reported that the
application of bovine derived xenograft and
bioresorbable collagen in patients with
periodontal defects resulted in significant
improvement, with reduction of periodontal
probing depth and clinical attachment levels
observed 1 year post-treatment.
Studies

35. T
issuee
n
g
i
n
e
e
r
e
dscaffoldsin periodontaltherapy

36. P
hysical forcesdetermineslongtermcomposition,
quality
,volumeoft
h
etissueconstruct....
- Mechanical forces as
regulators of tissue
growth, stem cell lineage
commitment and
differentiation, polarity,
motility, contractility &
apoptosis.

37. • Rigid substrate – Support high level isometric tension
• Flexible substrate – Cannot resist forces
- Turn off growth & differentiation
F
unctional e
ngin
e
e
ring....
-Theaim of improvingunderstanding of the rolethat mechanical
factorsplay in tissueregeneration.
-Mechanical signals askey regulators of the cellbehaviors required
for successfulengineered tissue growth.
-Bioreactorsarealready beingdeveloped that areableto
applylineage-specific mechanical signalsto populations of
stemcells

38. Competence factors (PDGF, FGF)
Progression factors
(IGF-I,
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 days
Biosignals

39. BMP’s
PDGF
BMP’s
IGF I,II
TGFβ
IGF I,II
TGFβ
Cell differentiation

40. T
ransform
ingGro
w
thfactors
• Majorgrowth factorsin bonematrix
• Superfamily of bonemorphogenicproteins
• A peptide synthesized & secretedin cell culture
• Increasesthe poolof committedosteoblasts
• Prevents long junctional epithelium formation
• Increasesosteoblasts chemotaxis

41. ContrellaMe
tal 1987
TGF -β
Smad
signalling
pathway
Migration
of
Osteogenic
progenitor
cells
Proliferate
Increases
osteoblasts
pool
Depends on dose & local
environment.
At high concentrations
of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Mae
dae
tal 2004

42. Miyazona K et al 2005
BMPs + BMPR1
BMPR2
Cell
Increases Increases MSX
cbfX1 gene gene exp
exp
Increases gene
exp of ALP, Epithelial &
osteocalcin mesenchymal
interaction
Matrix
formation &
mineralization
Bonem
o
rp
h
o
g
e
n
icpro
teins
•20 members.,Eg– BMP2,4,7
•BMP-2differentiationfactor
forbone& cartilageprecursor
cellsduringosteogenesis and
boneregeneration
•BMP5 through ActRIA
inhibitsmatrixsynthesis
• Induceectopicboneformation
•Driveendochondral ossification

43. BMPs acts on the pluripotent cells,
increases the committment &
differentiationof osteoblasts.
- Katagiriet al 1990
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenic
response
Franceschi e
tal 2000
BMPsshownformationof bonenodulesinvitro...
- ChenTLet al 1991
Limitations:
-No pdl fibre formation in
perpendicular direction and no acellular
cementum formation
- No role in osteoblast induction

44. Insulin likegrowthfactorsI &II
• Synthesized primarilyin liver and locally byosteoblasts.
• Mediate effect of systemic hormones(Eg., GH),cytokines
(IL-1α) & morphogens(BMPs) in boneformation & healing.
• Increasesproliferation effects of osteoblasts.
• Local regulatorof boneturnover
• IGF -I morepotent
• IGF –I upregulates theosteoblast associatedtranscription
factorOSTERIX but not cbfx1 & Runx2

45. IGF+ PDGFincreasesperiodontalregeneration
- LynchSEet al 1989
IGFI + BMP2 act synergistically onOSTERIX
- CelilAB et al 2003

46. FibroblastGrowthFactors
• Autocrine / Paracrineregulators of boneformation
• Stimulate chemotaxis, proliferation & matrix synthesis of
osteoblasts& osteoblast precursor.
• Play rolein angiogenesis& mesenchymal Cellmitogenesis
• Acceleratesfractureshealing
• FGF-2 most potent than FGF-1
- Contrellaet al 1988

47. Boneregeneration
GFs
IGF FGF
VEGF TGF PDGF
TNF-alpha , IL-1beta
Bone regeneration, remodelling & repair
BMPs,Cytokines, GF
Mesenchymal cells
Differentiation
Proliferation

48. Roleofvasculatureandneovascularization
in tissueengineering
• Cell survival…
– O2 Supply
– Nourishment
– Disposal of waste products
• Cells morethan approximately 200μmfromablood
supply arefound to beeither metabolicallyinactive or
necrotic.
• Tissue implantation volumes<2–3 mm3

49. V
asculature=1)vasculogenesis– d
e
n
o
v
oformation
2) angiogenesis– m
aturen
e
t
w
ork
Major angiogenic factors – FGF, PDGF and VEGF
Blood vessel
Angiogenic
Sprouting
VEGF
PDGF,Ang1

50. V
ascular endothelial growthfactor
• 6 proteins – VEGF A,B,C,D,E andplacental GF
• Splicedformslike VEGF 121, 165,189
• Primarytarget is endothelial cells
• Also for recruitment , survival and action of osteoblast&
osteoclast.
• Osteoblasts hasVEGF receptors& alsosecretesVEGF
• Mediates other GFs

51. V
ascular endothelial growthfactor
• Mousemodel;Fracture healing & cortical defect model;
- Street J et al 2002
• Major factor coupling osteogenesis +vasculogenesis
- GerberHPet al 2000

52. Plateletd
e
r
i
vedg
ro
w
t
hfacto
r
• 3 isoforms(AA,AB, BB); receptorsa& b;
• Chemotactic effect onosteoblast & Ct cells
- Hugheset al 1991
Increasescollagenasetranscription& increasesIL-6
expressionon osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000

53. Plateletd
e
r
i
vedg
ro
w
t
hfacto
r
• PDGF +βTricalciumphosphate GEM21S
rhPDGF +freeze dried boneallograft
Excellent results in intrabony defect
- Nevins M et al 2007

54. Applications

55. •Cell therapy

58. G
e
n
etherapy
“Genetherapyis theinsertionof genesinto an
individual's cellsand tissues to treat adisease,and
hereditarydiseasesin particular”.
Typicallyaimsto supplement adefectivemutant allele
with afunctional one.

60. • Transformation of ..
• Somatic cells
• Germ line cells (Sperm, Ova & stem Cells)
e
xviv
o
(wherecellsaremodifiedoutside
thebody andthentransplanted
backinagain)
inv
iv
o
(wheregenesare
changedincellsstill in
thebody.)

61. In Periodontics.....
A field of biomedicine.
• Not applied with success in periodontics
– Genetherapy Technology… far fromPerfect
– Periodontal disease…. Multifactorial…
– Genetic variations :
• multiplegenes,
• interactions between genesand theenvironment,

62. G
e
n
eenhancedTE...
• DeliveryofTherapeuticprotein-likegrowthfactor.
• -shortlife(afew hours).
• This is due to proteolytic breakdown and receptor
mediated exocytosisandsolubilityof thedeliveryvehicle

63. Studiesinp
e
riodontaltissu
ee
n
gine
ering
• Sankaranarayanan S et al 2013, in a 23-year female patient
with advanced periodontitis correlated with radiographic
evidence of severe horizontal bone loss extending up to the
apex of mandibular incisors; after debridement, the defect
was implanted with Autologous Bone Marrow Mononuclear
Cells impregnated in Thermo responsive Gelatin Polymer. In
6mo, PPD reduced to 2mm from 6mm & CAL 7mm from
13mm. At 36mo radiographic evidence in improvement of
vertical n horizontal height.

64. Studiesinperiodont
altissuee
n
g
i
n
e
e
r
i
n
g– R
e
vie
w
e
db
yElenaA.T
rofine
t
al 2013
• 7 case reports (for a total of 22 patients) where MSCs had been
applied clinically for infra-bony defects and furcation involvement
after chronic periodontitis in humans.
• The efficacy of human periodontal cell therapy by grafting
autologous stromal cells from gingiva or periodontal ligament
with a hydroxyapatite (HA) carrier has been assessed since 1992
(Feng and Hou, 1992; Feng et al., 1995, 2010; Hou et al., 2003).

65. Studiesinperiodont
altissuee
n
g
i
n
e
e
r
i
n
g– R
e
vie
w
e
db
yElenaA.T
rofine
t
al 2013
• Cultured mandibular periosteum-derived cell sheets with
PRP have also been used in the treatment of chronic
periodontitis without (Mizuno et al., 2010) or with HA in
patients suffering from advanced chronic periodontitis
(Okuda et al., 2009).
• A BMSC-PRP gel has also been used in an infrabony
periodontal defect and led to a 4 mm clinical attachment
gain (Yamada et al., 2006).
T
h
e
s
edatasuggestMSCsmayinduce efficient and
saf
eperiodontal regenerationin humans.

66. Futuredirectionsin periodontics...
1. GeneTherapeutics-Periodontal Vaccination
2. GeneticApproachto BiofilmAntibiotic
Resistance
3. An In vivo GeneTransfer byElectroporation
forAlveolar Remodeling.
4. Tight Adherence Gene for the Control of
Periodontal Disease Progression.
to Control
5. Antimicrobial Gene Therapy
DiseaseProgression
6. GeneTherapyto GrowNew Teeth
Tooth grown with urine
stem cells by a team from
the Guangzhou Institutes
of Biomedicine and
Health-
Cia et al

67. Cell s
h
e
e
te
n
g
ineering– Okanoe
tal 1993
• Temperature responsiveculture
dishes
• At 37 degreecelsius ;adhere&
proliferate
• Below 32 degreecelsiuscellsdetach
• Allows non-invasiveharvest of
cultured cellsasan intact monolayer
cellsheet includingdeposited ECM
• Enables direct transplantation
• 3D constructs suchasthick cardiac

68. Cell s
h
e
e
te
n
g
ineering

69. Artificial salivary gland
Baum BJ et al

70. Tissuee
n
g
i
n
e
e
ringfor dental implants–
Biomimeticmaterials
BiomimeticCa-Pcoatings
Both the superiormechanicalpropertiesof titanium and its alloys and
excellentbiocompatibility of Ca-Pmaterials.
Techniques
Radiofrequencymagnetronsputtering
Pulsed-laserdeposition
Ion-beamsputtering
Ion-beam-assisteddeposition
Electrophoretic techniques
BiomimeticT
echnique
GrowingaCa-Pthinlayeronmetalsorotherimplantmaterialsfroma
physiologicallyrelatedsupersaturated calcifyingsolution

71. BenefitsOfT
h
eBiomimeticApproachOverPlasma-
spraye
dHydroxyapatite…

72. Alb
umin
Plasma protein and plays afundamental role
in thetransport of otherproteins and
functional molecules in theblood.
Co-precipitatedinto theCa-Pcoating
KRAGH ETAL, 1990
As albumin canbind awide diversity of
ligands reversibly,with highaffinity, and
albumin microsphereshave beenused as
drugcarriers

73. BMP’
s
rhBMP-2incorporatedintoCa-P
coatings
Combinationof biomimeticCa-P
coatingsand osteoinductiveagents
canprovidesuperiorinductive
capability.

74. Bisposphonates
IncorporatedintoCa– Pcoatings
• Osteoclastinhibition
• Reduced boneturnover
• Increased bonemass
• Improvedmineralization.

75. • ToMasterthe art of recreatingfunctional viabletissuesin
thelaboratory…..translatetheknowledge….topopulation
at large
Promiseis GREAT…butchallengesexist
• Cost efficient…
• Availability
• Trainedstaff requirement
• Ethicalissues

76. Smile if you think
science is real .....