periodontal regeneration and advances in tissue engineering for regeneration of periodontium

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

2. Introduction
• See the hole ,fill it ,with anything
- Perio 2000 vol 50.
Principle Objective …
“To re-create functional, healthy tissues and organs
in order to replace diseased, dying, or dead tissues”

4. Periodontal Regeneration
• Appropriate cell types & Signals
• Local environment
– Recruitment of the right cells and
preventing the wrong cell
(Local Environment includes
Cementum matrix & C.E.J).
- Affects cell migration ,adhesion,
proliferation & differentiation.

5. Epithelial cells –
junctional
epithelium
Fibroblasts –
gingival
&periodontal
ligament fibres
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. To say periodontal regeneration has occurred 4
criteria should be fulfilled
• Functionalepithelialseal
• Newconnectivetissueattachment
• Acellularextrinsicfibercementum
• Alveolarboneheightrestored

7. Reasons for Failures in Periodontal
Regeneration technique
• Formation of a long junctional epithelium;
• Inadequate seal ;
• Wound closure
• Restriction of regeneration.
• Precise definition
• 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
(Narem R. et al.,1995 ; Rosso F. 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"
Langer and Vacanti
Tissue engineering

9. • Sixteenth century, Tagliacozzi 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. Key components
Scaffold
Progenitor cells
Biosignals

11. Diagrammatic representation of arteriovenous
shunt loop model in the rat – Mian R et al 2000
Artery
Venous graft
Hole of insertion
of vessel loop
Vein
Transparent chamber
Femoral vessels Leg

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

13. Stem Cells
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. Potency
The potency specifies the differentiation potential of the
stem cell.
• Totipotent stem cells
• Pluripotent stem cells
• Multipotent stem cells
• Unipotent stem cells

15. Key events in stem cell research
• 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis, ongoing stem cell activity in the brain;
their reports contradict Cajal's "no new neurons" dogma are largely
ignored
• 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
• 1968 - bone marrow transplant between two siblings successfully treats
SCID
• 1978 - haematopoietic stem cells are discovered in human cord blood
• 1981 - mouse embryonic stem cells are derived from the inner cell mass
• 1992 - neural stem cells are cultured in vitro as neurospheres
• 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo.

16. Key events in stem cell research
• 1997 - Leukemia was shown to originate from a
haematopoietic stem cell, the first direct evidence for cancer stem cells
• 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison.
• 2000s - several reports of adult stem cell plasticity are published
• 2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in
children's primary teeth
• 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes. The lines are
later shown to be fabricated
• July 19, 2006 - President George W. Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo

17. Types of Stem cells
Adult
stem
cells….
Embryonic
stem cells

20. in vitro
ADVANTAGES
The ability to
examine the material
as it is formed and to
perform specific
measurements prior
to implantation.
DISADVANTAGES
- The absence of a
physiologic & mechanical
environment during the
formation of tissue in vitro.
- Union of the implanted
tissues with the host organ
requires remodeling –
degradation and new tissue
formation at the interface
of implant with the host
tissue.

21. in vivo
ADVANTAGES
Tissue formation takes
place under the influence
of physiologic mechanical
environment.
Incorporation of the
tissues being formed with
the surrounding structures.
DISADVANTAGES
Regenerating tissues may
be dislodged or degraded
by the mechanical forces
normally acting at the site,
before the regenerating
tissue is fully formed and
incorporated.

22. I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976
-All cell lineages
- Mesenchymal fibroblasts cells
isolated
- Specific surface markers

23. Locally derived uncommitted cells
• Periodontal ligament stem cells
• Dental pulp stem cells
- Cell surface markers ( Gronthos & co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro.
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo

24. Periodontal ligament stem cells
• 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 compelling evidence : McCulloch and coworkers
1985,1987,1995,1996

25. • 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.

26. 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

27. Growth potential of pdl stem cells
• 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..

28. Characterization and origin of Periodontal
stem cells
• 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

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

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

31. Interactions between ECM & cell surafce recptors
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

32. Area code hypothesis...
• Hood L et al 1977
• The presence of a recognition system that guides cell
positioning
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
Cell adhesion
MMPs –
breakdown
and
remodellin
g of ECM...

33. Ideal properties
• 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.

34. 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.

35. Synthetic
• 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

36. Collagen sponge scaffold +
gelatin microspheres loaded
with FGF 2 in alveolar bone
defects - Nakahara et al. 2004
Vascularization, osteogenesis,
and recovery of periodontal
ligament function after 4 weeks.
Collagen sponges + periodontal
ligament cells = cementum had
uniformly regenerated along the
root surface of bony defects in
dog teeth
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

37. Tissue engineered scaffolds in periodontal therapy

38. Physical forces determines longterm composition,
quality, volume of the tissue construct ....
- Mechanical forces as
regulators of tissue
growth, stem cell lineage
commitment and
differentiation, polarity,
motility, contractility &
apoptosis.

39. Functional engineering....
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration.
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth.
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
• Rigid substrate – Support high level isometric tension
• Flexible substrate – Cannot resist forces
- Turn off growth & differentiation

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

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

42. Transforming Growth factors
• Major growth factors in bone matrix
• Superfamily of bone morphogenic proteins
• A peptide synthesized & secreted in cell culture
• Increases the pool of committed osteoblasts
• Prevents long junctional epithelium formation
• Increases osteoblasts chemotaxis

43. Contrella M et al 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
Maeda et al 2004

44. Miyazona K et al 2005
Cell
BMPs + BMPR1
BMPR2
Increases
cbfX1 gene
exp
Increases MSX
gene exp
Epithelial &
mesenchymal
interaction
Increases gene
exp of ALP,
osteocalcin
Matrix
formation &
mineralization
Bone morphogenic proteins
•20 members., Eg – BMP 2,4,7
•BMP-2 differentiation factor
for bone & cartilage precursor
cells during osteogenesis and
bone regeneration
• BMP 5 through ActRIA
inhibits matrix synthesis
• Induce ectopic bone formation
•Drive endochondral ossification

45. BMPs acts on the pluripotent cells,
increases the committment &
differentiation of osteoblasts.
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro...
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenic
response
Franceschi et al 2000
Limitations:
- No pdl fibre formation in
perpendicular direction and no acellular
cementum formation
- No role in osteoblast induction

46. Insulin like growth factors I & II
• Synthesized primarily in liver and locally by osteoblasts.
• Mediate effect of systemic hormones (Eg., GH), cytokines
(IL-1α) & morphogens (BMPs) in bone formation & healing.
• Increases proliferation effects of osteoblasts.
• Local regulator of bone turnover
• IGF -I more potent
• IGF –I upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 & Runx2

47. IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003

48. Fibroblast Growth Factors
• Autocrine / Paracrine regulators of bone formation
• Stimulate chemotaxis, proliferation & matrix synthesis of
osteoblasts & osteoblast precursor.
• Play role in angiogenesis & mesenchymal Cell mitogenesis
• Accelerates fractures healing
• FGF-2 most potent than FGF-1
- Contrella et al 1988

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

50. Role of vasculature and neovascularization
in tissue engineering
• Cell survival…
– O2 Supply
– Nourishment
– Disposal of waste products
• Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic.
• Tissue implantation volumes < 2–3 mm3

51. Vasculature = 1) vasculogenesis – denovo formation
2) angiogenesis – mature network
Major angiogenic factors – FGF, PDGF and VEGF
Blood vessel
Angiogenic
Sprouting
VEGF
PDGF,Ang1

52. Vascular endothelial growth factor
• 6 proteins – VEGF A,B,C,D,E and placental GF
• Spliced forms like VEGF 121, 165,189
• Primary target is endothelial cells
• Also for recruitment , survival and action of osteoblast &
osteoclast.
• Osteoblasts has VEGF receptors & also secretes VEGF
• Mediates other GFs

53. Vascular endothelial growth factor
• Mouse model ; Fracture healing & cortical defect model;
- Street J et al 2002
• Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000

54. Platelet derived growth factor
• 3 isoforms ( AA,AB, BB); receptors a & b;
• Chemotactic effect on osteoblast & Ct cells
- Hughes et al 1991
Increases collagenase transcription & increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000

55. Platelet derived growth factor
• PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007

56. Applications

57. •Cell therapy

60. Gene therapy
“Gene therapy is the insertion of genes into an
individual's cells and tissues to treat a disease, and
hereditary diseases in particular”.
Typically aims to supplement a defective mutant allele
with a functional one.

62. • Transformation of ..
• Somatic cells
• Germ line cells (Sperm, Ova & stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body.)

63. In Periodontics.....
A field of biomedicine.
• Not applied with success in periodontics
– Gene therapy Technology … far from Perfect
– Periodontal disease …. Multifactorial…
– Genetic variations :
• multiple genes,
• interactions between genes and the environment,

64. Gene enhanced TE...
Delivery of Therapeutic protein-like growth factor.
-short life (a few hours).
• This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle

65. Studies in periodontal tissue engineering
• 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.

66. Studies in periodontal tissue engineering –
Reviewed by Elena A. Trofin et 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).

67. Studies in periodontal tissue engineering –
Reviewed by Elena A. Trofin et 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).
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans.

68. Future directions in periodontics...
1. Gene Therapeutics-Periodontal Vaccination
2. Genetic Approach to Biofilm Antibiotic
Resistance
3. An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling.
4. Tight Adherence Gene for the Control of
Periodontal Disease Progression.
5. Antimicrobial Gene Therapy to Control
Disease Progression
6. Gene Therapy to Grow New Teeth
Tooth grown with urine
stem cells by a team from
the Guangzhou Institutes
of Biomedicine and
Health-
Cia et al

69. Cell sheet engineering – Okano et al 1993
• Temperature responsive culture
dishes
• At 37 degree celsius ; adhere &
proliferate
• Below 32 degree celsius cells detach
• Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
• Enables direct transplantation
• 3D constructs such as thick cardiac
muscle.

70. Cell sheet engineering

71. Artificial salivary gland
Baum BJ et al

72. Tissue engineering for dental implants –
Biomimetic materials
Biomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials.
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution

73. Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatite…

74. Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood.
Co-precipitated into the Ca-P coating
KRAGH ET AL, 1990
As albumin can bind a wide diversity of
ligands reversibly, with high affinity, and
albumin microspheres have been used as
drug carriers

75. BMP’s
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability.

76. Bisposphonates
Incorporated into Ca – P coatings
• Osteoclast inhibition
• Reduced bone turnover
• Increased bone mass
• Improved mineralization.

77. • To Master the art of recreating functional viable tissues in
the laboratory…..translate the knowledge ….to population
at large
Promise is GREAT…but challenges exist
• Cost efficient…
• Availability
• Trained staff requirement
• Ethical issues

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