The document discusses tissue engineering and its application in periodontal regeneration. It describes the key elements of tissue engineering which include progenitor/stem cells, scaffolds, and signaling molecules. It discusses various sources of progenitor cells for periodontal tissues, methods of scaffold fabrication, and growth factors used to enhance regeneration such as PDGF and BMPs. Studies evaluating the efficacy of different scaffold-growth factor combinations for achieving periodontal regeneration are also mentioned.

1. TISSUE
ENGINEERIN
G
Dr. BEENA VIJAYAN PARVATHY
3rd YEAR POST GRADUATE
Dept. of Periodontology and Oral
Implantology

2. CONTENTS
1. INTRODUCTION
2. KEY ELEMENTS IN TISSUE ENGINEERING
- Progenitor cells
- Scaffold
- Signalling molecules
3. DESIRED PROPERTIES AND WAYS TO ENHANCE THE
REGENERATIVE CAPACITY OF SCAFFOLDS
4. GENE THERAPY IN PERIODONTAL TISSUE ENGINEERING
5. RECENT DEVELOPMENTS
6. CRITICAL ANALYSIS OF PRESENT STATUS OF TISSUE
ENGINEERING FOR PERIODONTICS.
4. CONCLUSION
5. REFERENCES

3. INTRODUCTIO
N
DEFINITION
Tissue engineering is the branch of biology where tissues are produced
in culture by cells seeded (grown) in various porous absorbable
matrices by using biological principles.
Langer M et al; 1993
Tissue engineering is defined as the science of fabrication of new
tissues for replacement and regeneration of lost tissues or defined
tissues.
Baum et al; 2000

4. First proposed by Langer & Vacanti; 1993.
The primary aim of this therapy is to deliver biologically active
elements which get integrated into the host tissues and result in 3D
regeneration of the lost tissue which is structurally and functionally
similar to the tissue which was lost.
TE is a regenerative treatment of periodontal defects with an agent, or
procedure, requires that each functional stage of reconstruction be
grounded in a biologically directed process.
Bartold et al, 2000

5. KEY
ELEMENTS IN
TISSUE
ENGINEERING
CONDUCIVE
ENVIRONMENT
VASCULAR SUPPLY

6. APPROACHES USED TO REGENERATE TISSUES
EX VIVO APPROACH IN VIVO APPROACH
Tissue created in a lab by
culturing cells on a
biodegradable scaffold in
the presence of molecular
factors required for growth
and then transferred into
the body.
All components required
for regeneration are
placed in the tissue
defect and an
environment which is
conducive to maximum
regeneration is created to
achieve favorable
regeneration

7. PROGENITO
R
CELLS/STEM
CELLS
 These cells can differentiate into different types of end cells and can
form the desired structural components of the lost tissue.
Vats et al; 2002
 Criteria to achieve effective, long–lasting repair of damaged tissues:
1. Adequate number of cells must be produced to fill the defect.
2. Cells must be able to differentiate into desired phenotypes.
3. Cells must adopt appropriate 3D structural support / scaffold and
produce ECM.
4. Minimal associated biological risks.
5. Cells must integrate with native cells and overcome the risk of
immunological rejection.

8. SOURCE
AUTOGENIC
FROM ANOTHER
MEMBER OF SAME
SPECIES.
 ADV :
UNIFORMITY,
STANDARDISATION,
COST EFFECTIVE,
QUALITY CONTROL.
ALLOGENIC
 IDEAL
SOURCE
 FROM THE
PATIENT
 LOW IMMUNE
COMPLICATIONS
XENOGENIC
FROM OTHER
SPECIES
GREAT RISK OF
IMMUNOLOGICAL
ACTION.

9. PROGENITOR
CELL
PRODUCTION
IN
PERIODONTAL
TISSUES
PROGENITOR CELLS
PDL derived progenitor cells
Periosteal cells
Bone marrow derived MSCs
Adipose derived SCs
Gingival fibroblasts
PDL derived mesenchymal
stromal cells
1st evidence for presence of undifferentiated mesenchymal cells within periodontal tissues.
MuCulloch & co-workers; 1987

10. PDL
DERIVED
PROGENITO
R CELLS
 Capacity to produce cementum and periodontal ligament like
structures contributes periodontal tissue repair.
 PDL stem cells differentiates into cementoblast like cells and collagen
forming cells. Nakahara et al; 2004
 GTR  based on  these cells allowed to proliferate in the area of
periodontal defect to differentiate into cells required for regeneration.

11. PDL DERIVED
MESENCHYMA
L STROMAL
CELLS
Initially identified in adult bone marrow. Seo et al; 2004
 A clogenic clusters of adherent fibroblastic-like cells or fibroblastic
colony forming units with the potential to undergo extensive
proliferation.
 Have capacity to differentiate into different stromal cell lineages.
Friedstein et al; 1970,1976 & 1987
Consists of multipotency, clonogenic ability, high proliferation and the
expression of the putative stem cell marker STRO-1 & perivascular cell
marker CD146.

12. PERIOSTEAL
CELLS
 Differentiates into osteoblastic lineage, also express PDL related
genes.
Cells are clonogenic, displayed long tolomers and expressed markers
of mesenchymal stem cells (MSC’s).
De Bari et al; 2006

13. BONE
MARROW-
DERIVED
MESENCHYMA
L STEM CELLS
(BM-MSC’s)
Capable of producing bone, cartilage, adipose tissue, muscle and
periodontal tissues.
 PRP + BM-MSC’s  Periodontal regeneration.

14. ADIPOSE
DERIVED
STEM CELLS
(ADSC’s)
Used in periodontal regeneration.
Available in abundant.
 PRP + ADSC’s  Periodontal regeneration.

15. GINGIVAL
FIBROBLATS
 Root coverage  Recession  cell transplantation therapy using
gingival fibroblast.
Gingival fibroblast were seeded onto sponges of human Type I or III
recombinant collagen.
After culturing, vascular endothelial growth factor (VEGF) and
hepatocyte growth factor (HGF) released in culture media  ↑
fibroblast proliferation.

16. SCAFFOLD OR
SUPPORTING
MATRIX
 Scaffolds are natural or synthetic materials used to carry biologically
active molecules to the site of regeneration.
 Primary requirements:
1. Biocompatible
2. Biodegradable
3. Simple and predictable manufacturing process
4. Porous, mechanically stable and 3D structure.

17. SCAFFOLD
NATURALLY DERIVED SYNTHETICALLY DERIVED
Fibrin
Collagen
Chitosan
Alginate
Hyaluronic acid
Polyanhydrides
Polyorthoesters
Poly (α –
hydroxyesters)

18. ADVANTAGE OF SYNTHETICALLY DERIVED
Biocompatible
Biodegradable
Bioresorbable
Easily forms 3D structural matrices
 Poly (lactide-co-glycolide)(PLGA) copolymers  controlled
degradation behavior & mechanical properties can be adjusted
according to the requirements.
DISADVANTAGES OF SYNTHETICALLY DERIVED
Degrade  Produce acidic by products  hamper process of
regeneration.

19. SCAFFOLD
FABRICATION
Fiber bonding
Emulsion freeze drying
High pressure processing
Electrospinning
Rapid prototyping
Solvent casting / particulate
leaching
Thermally induced
phase separation
Gas foaming / particulate
leaching

20. FIBER
BONDING
PGA
PLA
Disadvantage:
Matrix porosity achieved cannot be precisely controlled.

21. EMULSION
FREEZE
DRYING
An emulsion solution containing a dispersed water phase and an
organic continuous phase is freeze dried.
It results in formation of a porous scaffold with various pore size and
interconnectivities.
Using this technique, up to 95% porous scaffold with a pore size, up
to 200 µm has been prepared.
Nuber et al; 1995

22. SOLVENT
CASTING /
PARTICULATE
LEACHING
This involves use of a water soluble porogen, such as salt.
In this technique, the polymer (PLLA or PLGA) is first dissolved in
chloroform or methylene chloride and then is casted onto a petri dish
filled with porogen.
Once the solvent evaporates, the polymer / salt composite is leached
in water for two days to remove the porogen.
The amount of porosity depends on the quantity of salt added and the
pore size depends on the crystal size of the salt particles.

23. HIGH
PRESSURE
PROCESSING
A gas such as CO2 is applied at high pressure to the dry polymer.
Results in the formation of a single phase polymer / gas solution.
After the formation of this single phase polymer / gas solution, the
pressure is reduced, which creates thermodynamic instability of the
dissolved CO2 and results in nucleation and growth of gas cells to
generate pores within the polymer matrix.
Mooney DJ et al; 1996

24. GAS FOAMING
/ PARTICULATE
LEACHING
A binary solution of PLA – solvent gel containing dispersed
ammonium bicarbonate salt particles.
The mixture was casted in a mold and subsequently immersed in hot
water.
Due to increased temperature, ammonia and CO2 gas are formed
along with the leaching out of ammonium bicarbonate particulates from
the solidifying polymer matrix.
Results in formation of a highly porous structure with high inter
connectivity.
Park et al; 2001

25. THERMALLY
INDUCED
PHASE
SEPARATION
This is by thermodynamic demixing of a homogenous polymer –
solvent solution into a polymer – rich phase and a polymer – poor
phase.
Liquid – liquid phase separation or emulsification / freeze – drying
method is used to separate the two phases.
The polymer solution is quenched below the freezing point of the
solvent and subsequently freeze dried.
Results in formation of a highly porous structure.

26. ELECTROSPINNIN
G
Most widely used method for preparation of nano fiber non – woven
matrices.
In this technique, a polymer solution is pumped at a constant rate
through a syringe with a small diameter needle that is connected to a
high – voltage source.
When this voltage source is turned on, an electric field is created
Under the strong electric field, electric charge overcomes the surface
tension of the polymer solution droplet.
Then a polymer jet is sprouted from the nozzle followed by solvent
evaporation which forms nanofibers.
Thus a highly porous 3D scaffold is formed.

27. RAPID
PROTOTYPING
A computer aided design ( CAD ) with pre – decided 3D architecture
is formed in a layer – by – layer manner with precise control over its
morphological characteristics.
 Most recent introduction.
Advantage:
Scaffold with a predetermined size, shape, porosity, chemical
composition and desired mechanical properties can be fabricated.

28. DESIRABLE PROPERTIES OF SCAFFOLDS USED FOR
PERIODONTAL REGENERATION
1. Cell-cell & cell-matrix interaction
2. Hold growth factors for desirable duration
3. Biocompatible
4. Allow proliferation
5. Should not induce environmental changes

29. RECENT
ADVANCES IN
SCAFFOLDS
MULTIPHASIC SCAFFOLDS

30. 3D-PRINTED SCAFFOLDS
c

31. GELS AND HYDROGELS
c

32. SMART SCAFFOLDS
-Biomimetic scaffolds & bionic smart scaffolds
-Immune sensitive smart scaffold
-Shape memory smart scaffold
-Electromechanical-stimulus smart scaffold

33. BIOMIMETIC
SCAFFOLDS &
BIONIC SMART
SCAFFOLDS
 Developed using inspiration from nature.
Elicit specified cellular responses mediated by interactions with
scaffold – tethered peptides, especially incorporating of cell – binding
peptides into biomaterials via chemical or physical modification.
Biomimetic porous poly (lactide-co-glycolide)(PLGA).
Mittal et al; 2010
 Scaffold fabricated by computer – generated design to mimic surface
morphology and pore size distribution. Thus regenerative potential
enhanced.

34. IMMUNE
SENSITIVE
SMART
SCAFFOLD
 Scaffold should have immunomodulatory properties, directing the
host response towards tolerance of the foreign scaffolds or regulating
immunological microenvironments to promote cell survival.
 IL-4 has been incorporated in the scaffolds to enable their
immunomodulatory capability.
Incorporation of nanofibrous heparin-modified gelatin microspheres
in the scaffold can spatiotemporally deliver the anti-inflammatory
cytokine IL-4 to polarize the proinflammatory M1 macrophages into an
anti-inflammatory M2 phenotype. It improves the osteogenic potential
of the scaffold.
Hu Z et al; 2018

35. SHAPE
MEMORY
SMART
SCAFFOLD
They can return from a deformed shape to their original shape by an
external stimulus, such as temperature change, an electric or magnetic
field and light.
 Scaffolds are fabricated by utilizing 3D and 4D printing technologies.
Field of minimally invasive surgical therapy (MIST).
The scaffold with small size is placed in the bone defect using
minimally invasive means with the least damage to host tissues. With time
the scaffold regains its actual size and precisely fills the bone defect.
BMP2-loaded shape-memory porous nanocomposite scaffold was
placed in bone defects in the rabbit model. The porous scaffold displayed
shape-memory recovery from the compressed pores of 33 μm in diameter
to recover its original porous shape of 169 μm in diameter, under both
invitro & invivo conditions. Promoted bone regeneration in mandibular
bone defects. Liu X et al; 2014

36. ELECTROMECHANIC
AL-STIMULUS
SMART SCAFFOLD
The piezoelectric property of certain materials can be utilized to
enhance the regenerative potential of the scaffolds.
The piezoelectric effect is the ability of a material to generate an
electric charge in response to applied mechanical stress.
Piezoelectric poly (vinylidene fluoride-trifluoroethylene) (PVDF-TrFE)
has been used to fabricate flexible, 3D fibrous scaffolds.
Damaraju SN et al; 2017

37. WAYS TO
ENHANCE THE
REGENERATIV
E CAPACITY OF
SCAFFOLDS
Addition of growth factors
•By soaking the scaffold in a solution of GF
•By encapsulation into scaffolds
•By covalent immobilization for controlled & extended release
•By incorporation into seeded cells via molecular & genetic
modification

38. SIGNALING
MOLECULES
 Secreted from various cells in response to stimulus and they act on
same, neighboring or distant cells to cause specific effects.
 GF stimulates synthesis of ECM by cells such as fibroblast,
osteoblast etc.
 Recombinant growth factor, commercial use:
•Platelet derived growth factor (PDGF; GEM 21)
•Bone morphogenic protein-2 (BMP-2; Infuse)
•Fibroblast growth factor (FGF-2)
GROWTH
FACTORS
BONE MORPHOGENETIC
PROTEIN

39. STUDIES
EVALUATING
EFFICACY OF
VARIOUS
SCAFFOLDS
USED WITH
SIGNALLING
MOLECULES IN
ACHIEVING
PERIODONTAL
REGENERATIO
N.

40. c

42. FDAAPPROVED MATRICES WITH GROWTH FACTORS
1. rhBMP-2 incorporated type-I bovine collagen sponge/ scaffold
(InFuse, Medtronic Sofamor Danek).
2. rhPDGF-BB with β-TCP scaffold
(GEM 21S, BioMimetic Therapeutics).
3. rhBMP-7 incorporated collagen sponge
(Osigraft, Stryker Biotech, Ontario, Canada).

43. FUNCTIONS OF
GROWTH
FACTORS
PDGF 
•Increases chemotaxis of PMNs & monocytes.
•Fibroblast proliferation, ECM synthesis.
• ↑ chemotaxis, proliferation of endothelial cells
•Fibroblast to myofibroblasts. Proliferation of mesenchymal progenitor
cells
TGF β
•Increases chemotaxis of PMNs & monocytes.
•Autocrine expression- generation of TNFα, IL1β, PDGF & chemokines
•Stimulates epithelial proliferation & migration.
•Fibroblast proliferation, ECM synthesis.
•Inhibits proteases & ↑ inhibitor production.
BMP 
•BMP2-4↑ mesenchymal progenitor cell migration.
•BMP7 ↑osteoblast differentiation.

44. GENE
THERAPY IN
PERIODONTAL
TISSUE
ENGINEERING
 The principle of gene therapy is to transfer desirable genes to the
target cell which then synthesizes a protein of interest.
 Transfer of gene to target cells;
The gene can be introduced;
VIRAL VECTORS NON – VIRAL VECTORS
Retroviruses
Adenoviruses
Adeno associated
viruses
Plasmid
DNA polymer
complexes
DIRECTLY INDIRECTLY

45. PROCEDURES
Harvesting the selected cell population
Expanding this population
Genetically transducing the genetic material
Re implanting cells in target area

46. RIBONUCLEIC
ACID
MEDIATED
SILENCING
Fire and Mello; 2006 discovered RNA interference gene silencing by
double stranded DNA.
It is a biological process in which RNA molecules inhibit the
expression of certain genes which are detrimental to the tissue
regeneration by causing the destruction of specific mRNA molecules.
 RNA interfernce is executed by 2 types of small RNA molecules :
1. microRNA
2. small interfering RNA
 RNAs are the direct products of genes, and these small RNAs can
bind to other specific messenger RNA molecules.

47. IMPLANTATION
OF LIVE CELLS
TO ACHIEVE
REGENERATIO
N
McGuire and Scheyer; 2007
Minimally invasive papilla priming procedure
To augment open interproximal spaces
Implanted autologous fibroblast
Results were significantly better in test sites than in placebo sites
Interdental papillary height Subject visual analog scale

48. Bowsma and D’souza; 2005
Expanded autologous fibroblast
Injected into periodontal pocket
Pocket depth reduction
Bowsma O, D’souza R, Meyerat BS. Treatment of deep
periodontal pockets with autologous fibroblasts or placebo.
J Dent Res 2005;84:107.

49. IMPLANTATION
OF TISSUE
ENGINEERED
HUMAN
FIBROBLAST
DERIVED
DERMAL
SUBSTITUTE
Human fibroblast derived dermal substitute ( HF-DDS ) is tissue
engineered living tissue derived from dermal fibroblasts.
One study compared the safety and effectiveness of a living HF-DDS
to a CTG for root coverage on Miller class I or II bilateral facial
recession.
The results of the study showed that HF-DDS may offer a potential
substitute to the CTG for root coverage.

50. APPLICATION
OF BI-LAYERED
CELL THERAPY
Tissue engineered bi-layered skin substitutes from human foreskin
 Type I collagen & viable allogenic human fibroblasts, keratinocytes
Substitute to palatal tissue.
 Momose et al;2002, estimated levels of vascular endothelial growth
factor(VEGF), transforming growth factor(TGFα & β), epidermal
growth factor(EGF) in human cultured gingival epithelial
sheets(HCGES).
 These GF’s influences the surrounding environment in favour of
healing.
Used in root coverage, increase width of keratinized gingiva and in
mucogingival surgeries.

51. CRITICAL
ANALYSIS OF
PRESENT
STATUS OF
TISSUE
ENGINEERING
IN
PERIODONTICS
Various growth factors act through intracellular signalling mechanism
once they attach to their corresponding surface receptors. Our
knowledge of these intracellular mechanisms is still incomplete.
Presently , we have insufficient data for the authentication of clinical
safety and effectiveness of various newer regenerative techniques by
tissue enginering.
The exact mechanism by which the growth factors enhance
periodontal regeneration yet remains to be proven in vivo. Although
tritiated thymidine and proline labelling studies would yield valuable
information regarding in vitro effects of PGDF/IGF-1, more research is
required in this field.

52.  Cost effective application, and easy availability still need to be sorted
out.
Ideally, once delivered at the site of interest, the growth factors should
act on their target cells to produce desired effects. However, there are
many mechanisms which neutralize or deactivate these growth factors
when placed in the biological environment.
 Cell culture media of xenogenic products always carry a risk of
disease transmission. Newer identification techniques are required to
authenticate the safety of xenogenic products.
There are always possibilities of immune rejection of the implanted
cell line when allogenic and xenogenic sources of the cell line are used.
Need to be well investigated for any immunological reaction.

54. REFERENCES
1.Baumm BJ, Mooney DJ. The impact of tissue engineering on
dentistry. J Am Dent Assoc 2000 ; 131( 3) : 309 – 18.
2.Langer R. tissue engineering. Science J 1993 : 260.
3.Advanced reconstructive technologies for periodontal tissue repair.
Perio 2000.
4.A novel approach to periodontal tissue regeneration with
mesenchymal stem cells and platelet rich plasma using tissue
engineering technology. A clinical case report 2006.
5.Yamamiya, J. Periodontal 2008 , Tissue engineered cultured
periosteum used with platelet rich plasma and hydroxyapatite in treating
human osseous defects.
6.Nakahara T et al, in situ tissue engineering of periodontal tissues by
seeding with periodontal ligament derived cells, 2004.
7.Bartold PM, Xiao Y, Principles and applications of cell delivery
systems for periodontal regeneration, Perio 2000 – 2006.