7. Ð Term TE covers a broad range of applications.
Ð In practice-term is closely associated with
applications that repair or replace portions of or
whole tissues i.e.
Bone
cartilage
blood vessels
bladder
Skin
Ð Tissues involved require certain mechanical &
structural properties for proper functioning.
7
9. TISSUE ENGINEERING
In 1987, Term ―tissue engineering‖ was coined
at a National Science Foundation (N.S.F.)
bioengineering meeting in Washington D.C
VACANTI & LANGER,
―A combination of the principles & methods of
life sciences with that of engineering, to develop
materials & methods to repair damaged or diseased
tissues, & to create entire tissue replacements‖
9
10. DEFINITION
SHALAK & FOX 1988,
―The application of principles & methods of
engineering & life sciences, to obtain a
fundamental understanding of structural &
functional relationships in novel & pathological
mammalian tissues, & the development of
biological substitutes to restore, maintain or
improve tissue function‖
10
11. HISTORICAL BACKGROUND
In 1970 W.T. Green, an orthopedic surgeon
conducted 1st research related to TE.
suggested that by
implanting chondrocyte cells into spicule of
bone, where cell multiplication & growth of
bone continues →cartilage formation
11
12. In the Mid-1980’s Dr. Vacanti and Dr. Langer
devised a method that would attempt to create
scaffoldings for cell delivery instead of using
naturally occurring scaffoldings that could not be
replicated.
12
13. In 1994, TES was founded by Charles & Vacanti
officially in Boston.
By 2005, TERMIS which included both Asian &
European Societies, was created.
13
14. NEED FOR TISSUE ENGINEERING
Tissue engineering holds promise of producing
better organs for transplant. Using tissue
engineering techniques & gene therapy it may be
possible to correct many otherwise incurable
genetic defects.
A major goal of tissue engineering is in-vitro
construction of transplantable vital tissue.
Artificial tissues can revolutionize healthcare by
providing a supply of soft & hard CT on demand.
14
15. Major shortcoming autografts & allografts in achieving
regeneration
humans don’t have significant stores of excess tissue for
transplantation
15
17. Successful tissue engineering requires interplay
among three components:
Implanted & cultured cells that will create new
tissue;
Biomaterial to act as scaffold or matrix to hold
cells;
Biological signaling molecules that instruct cells
to form desired tissue type.
17
23. IN VITRO
Construction in laboratory of vital tissue & its
subsequent implantation into host body.
Advantage is ability to examine tissues as
they are formed, & to perform specific tissue
measurements.
23
24. By in-vitro TE of tissues such as bone, need
for recruitment of specific cells to site is
negotiated & predictability of regeneration is
enhanced,
overcoming many of limitations with
conventional therapies.
Disadvantage is absence of a physiologic
environment
Implanted tissue has to be incorporated with 24
25. IN VIVO
Indicates obvious advantage of tissue regeneration
in-vivo in which incorporation occurs as tissues are
formed.
This has formed basis for tissue engineering,
which now includes implantation of porous matrices,
seeded with appropriate cells & signalling molecules, to
facilitate tissue regeneration in-vivo.
25
26. Disadvantage of in-vivo approach
regenerating tissues may get dislodged or
degraded by mechanical forces acting normally at
site, before regenerated tissue is fully formed &
incorporated
26
28. ORIGIN OF CELLS
Osteogenic cells could be obtained through an
atraumatic biopsy & amplified in an appropriate 3-D
carrier in-vitro.
28
29. MODES OF SUPPLY
There are two modes for supplying exogenous cells
into defect:
Cell seeding
Cell suspension
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.
29
30. SOURCES
Autologous cells (the host’s own cells)
Allogenic cells (cells from a donor)
Xenogenic cells (cells from a different species)
Stem cells: either allogenic (fetal or adult
derived) or autologous (adult derived).
30
31. Autologous cells are obtained from same
individual to which they will be re-implanted.
Have fewest problems with rejection &
pathogen transmission, however in some
cases might not be available.
Example genetic disease suitable
autologous cells are not available.
These cells can differentiate into
a variety of tissue types, including
bone, cartilage, fat, & nerve.
31
32. Allogeneic cells come
from body of a donor
of same species.
Employment of
dermal fibroblasts fro
m human foreskin
has been
demonstrated to be
immunologically safe
& thus a viable
choice for TE of skin. 32
33. Xenogenic cells are
these isolated from
individuals of another
species. In particular
animal cells have been
used quite extensively
in experiments aimed
at construction of CV
implants.
33
34. STEM CELLS
Undifferentiated cells with ability to divide in culture
& give rise to different forms of specialized cells.
Characteristic Features:
They are capable of dividing & renewing themselves for
long periods
They are unspecialized
They can give rise to specialized cell types.
34
36. Adult stem cells Also known as somatic (from Greek "of
the body") stem cells & germline (giving rise to gametes)
stem cells, they can be found in children, as well as adults.
Pluripotent adult stem cells are rare & generally small in
number but can be found in a number of tissues including
umbilical cord blood
Most adult stem cells are
lineage-restricted & are generally
referred to by their tissue origin
36
37. Embryonic stem cell lines are cultures of cells
derived from epiblast tissue of inner cell mass of
a blastocyst or earlier morula stage embryos —
approximately 4 to 5 days old in humans &
consisting of 50–150 cells. ES cells are pluripotent
& give rise during development to all derivatives of
3 primary germ layers:
ectoderm,
endoderm &
mesoderm.
37
38. Based on potency the cells are divided into:
1. Totipotent cells.
2. Pluripotent cells.
3. Multipotent cells.
4. Oligopotent cells.
5. Unipotent cells.
38
39. Totipotent stem cells can differentiate into
embryonic & extraembryonic cell types. Such cells
can construct a complete, viable organism. These
cells are produced from fusion of an egg & sperm
cell. Eg: Fertilized egg
39
40. Pluripotent stem cells are descendants of
totipotent cells & can differentiate into nearly all
cells, but cannot give rise to an entire organism. i.e.
cells derived from any of three germ layers
Multipotent stem cells give rise to a limited range
of cells within a tissue type. Eg: Hematopoietic
stem cells.
40
41. Oligopotent stem cells can differentiate into only
a few cells, such as lymphoid or myeloid stem cells.
Unipotent cells can produce only one cell type,
their own, but have the property of self-renewal,
which distinguishes them from non-stem cells. E.g.
muscle stem cells.
41
42. APPLICATION
Cell Replacement Therapies
Cells could be stimulated to develop into
specialized cells that represent renewable
sources of cells & tissue for transplantation.
Cell replacement therapy could treat injuries &
various genetic & degenerative conditions
including muscular dystrophies, retinal
degeneration, Alzheimer disease, Parkinson's
disease, arthritis, diabetes, spinal cord injuries,
& blood disorders such as hemophilia.
42
44. SCAFFOLDS
Used to
guide
organization,
Growth & differentiation of cells in process of forming
functional tissue
provide both physical & chemical signals.
Tissues are composed of
cells,
insoluble extracellular matrix (E.C.M.)
soluble molecules that serve as regulators of cell
function.
44
45. E.C.M. usually composed of 3 components:
Collagen
Glycoprotein
Proteoglycan
The E.C.M. is important for
Growth
Function - various cell types involved.
45
47. NON-ABSORBABLE
SYNTHETIC CERAMICS
Implemented as matrix materials for facilitating regeneration in-vivo
(Bucholtz et al 1987). 2 most widely used forms are:
Tricalcium phosphate
Hydroxyapatite.
1. Tricalcium Phoshphate:
Porous form of calcium phosphate
ß-TCP
Problem -physiochemical dissolution after implantation
2. Synthetic Hydroxyapatite:
development - second form of bioceramic.
Rationale - mineral naturally occurring in bone is
hydroxyapatite.
47
48. NON-ABSORBABLE
SYNTHETIC POLYMERS
PTFE – synthetic
fluoropolymer of tetrafluoroethylene that finds
numerous applications. well known brand
name of PTFE is Teflon by DuPont Co.
48
49. ABSORBABLE
SYNTHETIC POLYMERS
degradation by hydrolysis
Polyglycolic acid - degrades fast
Polylactic acid (L-lactide) - most stable in-vitro
Thus, modification of poly (L-lactide) by cross-
linking or addition of D-lactide more rapid
degradation, thus diminishing poly L-lactide
disadvantage of slow degradation.
polyglactin 910, a co-polymer of glycolide and L-
lactide – 90/10 molar ratio
49
50. ABSORBABLE
NATURAL POLYMERS
Collagen - protein with 3 polypeptide chains, known
as α-chains, each containing at least 1 stretch of
repeating AA sequence
Collagen constitutes almost 1/3 of all protein in
body, & accounts for almost 60% of gingival
connective tissue & 90% of total protein in bone.
50
51. Collagen - medical devices,
derived from animal sources,- bovine skin, tendon,
intestine or sheep intestine.
Collagen based sutures & hemostatic sponges
have also been used.
Resorbable collagen barriers have been used
clinically for G.T.R. procedures, although their
combination with biologic modifiers has not been
explored.
Also, absorbable collagen sponge (ACS) has
been used as a carrier for rhBMP-2
51
52. ABSORBABLE
NATURAL MINERALS
HA skeleton (Bio-Oss®, Osteograf®) -
retains microporous & macroporous
structure of cortical & cancellous bone.
remaining after chemical or low heat
extraction of the organic component.
Usually bovine bone mineral is used
Currently available - deproteinated, which
supports cell-mediated resorption.
52
54. SIGNALLING MOLECULES
Signalling molecules or biologic modifiers -
materials or proteins & factors that have
potential to alter key cellular events in host
tissue, by stimulating or regulating the wound
healing process.
54
56. CLASSIFICATION
3 groups
1. Growth & Differentiation Factors
2. Extracellular Matrix Proteins & Attachment Factors
3. Mediators of Bone Metabolism
56
57. GROWTH AND DIFFERENTIATION FACTORS
Growth factors - play important role in
regeneration are:
1) Platelet derived growth factor (P.D.G.F.),
2) Insulin-like growth factor (I.G.F.),
3) Transforming Growth Factor- β (T.G.F.-β),
4) Fibroblast Growth Factor,
5) Bone Morphogenetic Proteins (B.M.P.s).
57
58. PLATELET DERIVED GROWTH FACTOR
CHEMISTRY: 2 disulphide bonded poly-peptide
chains that encoded by 2 different genes-P.D.G.F.-
A & P.D.G.F.-B.
FORMS: exist either as
heterodimer (AB) or
homodimer (AA, AB).
3 isoforms of PDGF have unique binding properties
for PDGF receptor sub-units, α & β, found on cell
membrane.
58
59. PRODUCTION: Several cell types produce
PDGF, including
Degranulating platelets,
Smooth muscle cells,
Fibroblasts,
Endothelial cells,
Macrophages & keratinocytes.
59
60. RECOMBINANT BMP-2 PRODUCTION
Recombinant proteins are produced from one of
several cellular expression systems:
Bacteria,
Insect cells or mammalian cells.
rh BMP-2 is produced using mammalian cell
expression system, which allows for proficient
execution of post-translational modifications that
are present in human BMPs.
60
61. Chinese Hamster Ovary (CHO) cells are host of
choice. Because mammalian cells synthesize a
variety of GF, they are capable of synthesizing &
secreting active BMP.
Includes many steps:
Synthesizing of precursor polypeptide chains.
Correct refolding & demineralization of these chains,
Glycosylation of protein.
61
62. Ð Protein is then secreted
out of cell into
conditioned medium,
in process of which
propeptide is removed from
mature portion of protein at
specific AA sequences.
62
63. INSULIN-LIKE GROWTH FACTORS (IGF-I,II):
Peptide growth factors
with biochemical &
functional similarities
to insulin.
Bone cells produce &
respond to IGF’s, and
bone is a storage
house for these factors
in their inactive form.
63
64. TRANSFORMING GROWTH FACTOR-β:
Multifactorial growth factor, structurally related to
B.M.P.s, but functionally quite different.
Chemotactic for bone cells, & may increase or decrease
their proliferation depending upon the differentiation
state of the cells, culture conditions and concentration of
TGF-β applied.
In-vivo, produces new cartilage and / or bone, if injected
in proximity to bone; however, it does not induce new
bone formation when implanted
away from a bony site.
64
65. BONE MORPHOGENETIC PROTEINS (BMPS):
Urist in 1965, reported that protein extracts
from bone, implanted into animals at non-
bone sites induced formation of new cartilage
& bone tissue.
65
66. MODES OF PREPARATION:
2 modes of preparation have been used:
Preparationsderived from bovine or human
bone, which contains complex mixture of BMP
molecules & possibly other factors & proteins
RECOMBINANT DNA METHODS-
when recombined with DNA of cloning vector, can
be replicated, transcribed & translated. Used for
production of (rh BMP-2) & (rh BMP-7).
66
67. rh BMP2 PRODUCTION
cDNA CODING FOR rh BMP-2
TRANSFECTED INTO HOST CELL
(CHO CELL)
rh BMP-2 SECRETED
STORED IN ALIQUOTS & FROZEN
67
68. PUT IN GROWTH MEDIUM &
HARVESTED
rh BMP-2 REMOVED BY FILTRATION
PURIFIED BY COLUMN
CHROMATOGRAPHY
PLACED IN VIALS & LYOPHILIZED
68
69. MEDIATORS OF BONE FORMATION
Several agents which affects the growth of bone:
PROSTAGLANDINS:
Result of cyclo-oxygenation of precursors
derived
from arachnoid acid. Found - variety of tissues. Effect
varies
considerably from stimulating inflammation & bone
resorption
to enhance bone formation
GLUCOCORTICOIDS:
Such as dexamethasone have prostaglandins, complex
direct & indirect effects on bone formation. Chronic
glucocorticoids administration results in bone loss, through
69
70. BISPHOSPHONATES:
A class of pharmacuetical agents, which
are structurally similar to pyrophosphates, natural
product of human metabolism.
Bisphosphonates binds to HA crystal of
bone & prevent their growth & dissolution
CLASSIFIED AS:
1st Generation : alkyl side chains
Eg: Endronate
2nd Generation : amino terminal grp.
Eg: Alendronate & Pamidronate
3rd Generation : cyclic side chains BISPHOSPHONATES
Eg: Risedronate
PYROPHOSPHATES
70
71. FIBROBLAST GROWTH FACTORS:
Family of at least 9 related gene products of which
2 major members are a-FGF or FGF-1 & b-FGF or
FGF-2.
Stimulate endothelial cells & PDL cell migration
& proliferation, as well as stimulation of bone cell
replication.
b-FGF is more potent than a-FGF & may act via
stimulation of other growth factors like TGF-β.
71
72. PLATELET RICH PLASMA
PDGF & TGF-β are well-established wound healing
―hormones‖.
One of highest concentrations of PDGF & TGF-β in
body are found within α-granules of blood platelets
Thus, concentrating platelets would result in
concentration of growth factors, enhancing wound
healing on application.
72
73. PROCESSING OF P.R.P.
Autologous platelet rich plasma (PRP) was
developed in the 1970’s as a by-product of multiple
component apheresis.
Today, there are 3 main techniques available for
procurement of PRP:
Procurement from Procurement on
Apheresis
one unit blood a small scale
73
74. APHERESIS
The process of apheresis basically involves
removal of whole blood from a patient or donor.
Within an instrument that is essentially designed as
centrifuge components of whole blood are
separated.
One of components is then
withdrawn & remaining components
are re-transfused into patient or donor.
74
75. PROCUREMENT FROM ONE UNIT BLOOD:
Uses one unit (350 ml) of the patient’s blood, but
instead of using an apheresis apparatus, it uses a
temperature-controlled centrifuge (cold
centrifuge).
Whole blood is obtained in a transfusion bag &
subjected to a low spin cycle of 1100 rpm for 15
minutes, which results in separation of 3 basic
fractions
75
76. PROCUREMENT ON A SMALL SCALE
Recent studies focussed on using minimal amount
of blood (10-50 ml) depending upon procedure
involved, & common laboratory centrifuge for
procurement of PRP.
This procedure uses double-spin centrifugation
(2,400 rpm for 10 minutes, & then after discarding
RBC fraction, 3,600 rpm for 15 minutes), & 3
components are obtained in test-tube.
76
77. ADVANTAGES OF USE OF AUTOLOGOUS P.R.P.
Safe as it is autologous preparation.
Promotes adhesiveness & tensile strength for clot
stabilization.
Biologically acceptable.
Contains growth factors (PDGF & TGF-β) released
by platelets.
77
78. Promotes angiogenesis.
Haemostatic properties.
Dense fibrin net that is highly osteoconductive.
High concentrations of leukocytes, which act as
―autologous antibiotic‖, reducing risk of infection.
78
80. GENE THERAPY
A problem with current delivery of growth factors to
wounds is extremely short half-lives of these
factors. This can be attributed to:
Proteolytic breakdown.
Receptor mediated endocytosis.
Solubility of delivery vehicle.
80
81. GENE EXPRESSION & PROTEIN SYNTHESIS
Genes are specific portions of DNA that code for
proteins. Their role in protein synthesis can be
illustrated as follows:
Activation of transcription via cell surface receptors.
Transcription of DNA code into mRNA.
Processing of mRNA in preparation for
transportation to cytoplasm.
Transport of mRNA to cytoplasm.
81
82. RNA translation & peptide synthesis.
Polypeptide elongation.
Post-translational modifications.
Transport to & across cell membrane.
At each stage of gene expression, there is an
opportunity for control & regulation of protein
synthesis.
82
83. GENE TRANSFER
2 general ways to transfer genes:
Virus mediated vectors: - ex-vivo approach
- in-vivo
approach
Naked DNA using Plasmids.
Transduction (i.e. transfer of genetic
fragment) to appropriate target cells (i.e.
osteoblasts) represents first critical step in
gene therapy.
83
84. VIRAL METHODS
• Introduce RNA with two enzymes- reverse transcriptase & integrase
• Enable productin of DNS from RNA – latter add DNA copy to target cell
DNA
• DNA is transferred into target cell nucleus, but not integrated with host
DNA.
• Infects dividing & non-dividing cells
• Small viruses with single stranded DNA that cause no human disease.
• Infects dividing & non-dividing cells
84
85. NON VIRAL METHODS
Micro seeding • Direct injection of therapeutic DNA into target cells using
a gene gun
gene therapy
Cationic • Creation of artificial lipid spheres with an aqueous core
• Carries therapeutic DNA, capable of passing DNA
Liposomes through target cell membrane
Macromolecular • Therapeutic DNA gets inside target cells by chemically
linked DNA to molecule that bind to special cell receptor
Conjugate
Gene Activated • Delivers naked DNA via polymer matrix sponges.
Matrices
85
86. SOFT TISSUE AUGMENTATION
Most commonly used applications of tissue
engineering is in field of dermatology, where
possibility of obtaining a large amount of dermal-
epidermal tissue from a small portion of skin of
same patient in a short period of time, has allowed
treatment of extensive burns.
86
88. Future developments in fields of molecular & cell
biology, developmental biology & tissue engineering,
will have significant impact on managing anatomic
changes due to disease process.
88
89. REFERENCES
Vacanti, Charles A. "The history of tissue
engineering." Journal of Cellular and Molecular
Medicine 10 (2006): 569-76.
Lynch SE, Genco RJ, Marx RE. Tissue Engineering:
applications in maxillofacial surgery and periodontics.
Tissue engineering - Wikipedia, the free
encyclopedia.
Langer R, Vacanti JP (May 1993). "Tissue
engineering". Science 260 (5110): 920–
6. doi:10.1126/science.8493529.
Stem cell - Wikipedia, the free encyclopedia.
Stem Cells: General Features and Characteristics.
Hongxiang Hui.
89
