This document provides an overview of principles of tissue engineering. It discusses why tissue engineering is needed due to limited organ transplantation availability. Tissue engineering uses regenerative medicine approaches including cell therapies, biomaterials, and tissue engineering to repair or replace damaged tissues. Various cell sources for therapy are described, including stem cells (embryonic, adult, perinatal), somatic cell nuclear transfer, and induced pluripotent stem cells. Biomaterials are discussed that can be used as scaffolds to support cell growth. The importance of vascularization for tissue volumes over 3mm is also highlighted.
2. Why?
Organ transplantation remains a mainstay of treatment for
patients with severely compromised organ function.
Despite initiatives to increase the availability of transplant
organs, however, the number of patients in need of treatment far
exceeds the organ supply, and this shortfall is expected to
worsen as the global population ages.
One alternative treatment approach is regenerative medicine.
3. What?
“An interdisciplinary field of research and clinical applications
focused on repair, replacement or regeneration of cells, tissue
or organs to restore impaired function from any cause(ie.
Congenital defects, disease, trauma, ageing)”
Combines converging technological approaches, both existing
and newly emerging, moving it beyond traditional
transplantation and replacement therapies.
Approaches often aim to stimulate and support the body’s own
self healing capacity
4. How?
Approaches may include:
Use of soluble molecules
Gene therapy
Stem and progenitor cell therapy
Tissue engineering
Reprogramming of cell and tissue types
5. Regenerative Medicine: Strategies for Tissue
and Organ Reconstitution
Cell-based therapies
Biomaterial based therapies
The use of biomaterials (scaffolds) alone, wherein the body’s
natural ability to regenerate is used to orient or direct new tissue
growth)
Combined (tissue engineering)
The use of scaffolds seeded with cells to create tissue substitutes.
6. Sources of Cells for Therapy
A. Stem Cells
B. Native Targeted Progenitor Cells
C. Therapeutic Cloning (Somatic Cell Nuclear Transfer)
D. Reprogramming (Induced Pluripotent Stem Cells)
7. A] Stem Cells
Defined as having three important properties:
Ability to self-renew,
Ability to differentiate into a number of different cell types,
Ability to easily form clonal populations
Autologous, Allogenic
8. Stem Cells
Three broad categories
Embryonic stem cells
Fetal and neonatal amniotic fluid and placenta
Adult stem cells
9. 1)Embryonic Stem Cells
Human embryonic stem cell-
1981,Martin
Pluripotent cells- skin to neurons
form embryoid bodies
form teratomas in vivo
Found in the inner cell mass of
the human embryo
Able to differentiate into all cells
of the human body, excluding
placental cells
( Only cells from the morula are
totipotent )
maintain undifferentiated state
for at least 80 passages
10. Embryonic Stem Cells
Ideal resource for regenerative medicine
Ability to self-renew indefinitely
Ability to differentiate into cells from all three embryonic germ
layers
Limited clinical application : Allogenic
Ethical issues : results in destruction of embryos
11. Embryonic Stem Cells
Alternate techniques without destroying embryos
single-cell embryo biopsy
Arrested embryos
Altered nuclear transfer
12. 2)Perinatal Stem Cells
Umbilical cord blood
collected at the time of birth
more immature than adult bone marrow stem cells
autologous and allogeneic cell therapy
mostly used for hematopoietic applications
13. Perinatal Stem Cells
Wharton jelly
surrounds the cord
mesenchymal in origin
limited multipotentiality
not been used clinically
14. Perinatal Stem Cells
Amniotic fluid and placenta
Capable of extensive self-renewal
Give rise to cells from all three germ layers
Properties somewhere between those of embryonic and adult stem
cell
Do not form teratomas
For self-use, avoid the problems of rejection
Obtained either from
Amniocentesis or
Chorionic villous sampling in the developing fetus or
From the placenta at the time of birth.
15. 3)Adult Stem Cells
Many types of adult stem cells have been identified in organs
throughout the body and are thought to serve as the primary
repair entities for their corresponding organs
Limited clinical application :
Some cells have very low proliferative capacity in vitro
Functionality is reduced after the cells are cultivated
Isolation of cells has also been problematic
16. Adult Stem Cells
Mesenchymal stem cell
Multipotent adult
progenitor cell
Derived from bone
marrow stroma
Can differentiate in vitro
into numerous tissue
types
17. Adult Stem Cells
Hematopoietic stem cells
Best understood
Used for decades for hematopoietic disorders
Adipose-derived stem cells
Could give rise to multiple lineages
Differentiation into urologic cells
Used experimentally to improve bladder function
Urine-derived stem cells have also been proposed for
genitourinary reconstruction
18. B] Native Targeted Progenitor Cells
Tissue specific unipotent cells derived from most organ
Already programmed to become the cell type needed, without
any extra-lineage differentiation
obtained from the specific organ to be regenerated, expanded,
and used in the same patient without rejection
19. Native Targeted Progenitor Cells
Exploring the conditions that promote differentiation and/or self-
renewal, it has been possible to overcome some of the obstacles that
limit cell expansion in vitro
donor tissue is dissociated into individual cells
which are either implanted directly into the host or expanded in culture,
attached to a support matrix, and re-implanted after expansion.
20. Native Targeted Progenitor Cells
System of urothelial cell harvesting was developed that does not
use any enzymes or serum.
Has a large expansion potential
Possible to expand a urothelial strain from a single specimen
(1cm2)to one covering a surface area of one football field(4202m2)
within 8 weeks.
21. Native Targeted Progenitor Cells
Bladder, ureter, and renal pelvis cells can equally be harvested,
cultured, and expanded
Not all human cells can be grown or expanded in vitro e.g. Liver,
nerve, and pancreas
22. C] Somatic Cell Nuclear Transfer
Removal of an oocyte nucleus in culture, followed by its
replacement with a nucleus derived from a somatic cell obtained
from a patient
Two types of cloning
Reproductive cloning
Therapeutic cloning
Both involve the insertion of donor DNA into an enucleated
oocyte to generate an embryo that has identical genetic material
to its DNA source.
23. Somatic Cell Nuclear Transfer
Reproductive cloning
Reproductive cloning:
the embryo is implanted into the uterus
of a pseudo-pregnant female to produce
an infant that is a clone of the donor
Eg : birth of a sheep named Dolly in
1997
24. Somatic Cell Nuclear Transfer
Therapeutic cloning
Embryo is used to generate blastocysts that are explanted and
grown in culture rather than in utero
Embryonic stem cell lines can then be derived from blastocysts,
which are allowed to grow only up to a 100-cell stage
At this time the inner cell mass is isolated and cultured
Resulting in embryonic stem cells that are genetically identical
to the patient
25. Somatic Cell Nuclear Transfer
Therapeutic cloning
Pluripotent nuclear transferred embryonic stem cells have been
derived from
fibroblasts,
lymphocytes,
olfactory neurons
perfectly matched to the patient’s immune system and no
immunosuppressants would be required
However, mitochondrial DNA contained in the oocyte could lead
to immunogenicity after transplantation
26. Somatic Cell Nuclear Transfer
Therapeutic cloning
Ethical considerations regarding the potential of the resulting
embryos to develop into cloned embryos
Limitations that require further improvement
not been shown to work in humans to date
27. D] Reprogramming
(Induced Pluripotent Stem Cells)
Involves de-differentiation of adult somatic cells to produce
patient-specific pluripotent stem cells, eliminating the need to
create embryos
genetically identical to the somatic cells and would not be
rejected
iPS cells possessed the characteristics of
self-renewing embryonic stem cells
expressed genes specific for embryonic stem cells
generated embryoid bodies in vitro
teratomas in vivo
28. Reprogramming
(Induced Pluripotent Stem Cells)
Generated human iPS cells are similar to hESCs in terms of
morphology, proliferation, gene expression, surface markers, and
teratoma formation
Cells have shown great promise in the understanding of human
disease, as well as the use of these cells for therapy.
Like embryonic stem cells, the iPS cells also form teratomas, and
this has limited their therapeutic potential.
30. Biomaterials and Vascularization for
Genitourinary Regenerative Medicine
Biomaterials function as an artificial ECM and elicit biologic and
mechanical functions of native ECM
Biomaterials facilitate :
localization and delivery of cells and/or bioactive factors
define a 3D space for formation of new tissues with appropriate
structure
guide development of new tissues with appropriate function
31. Design and Selection of Biomaterials
Must be capable of controlling the structure and function of the
engineered tissue in a predesigned manner by interacting with
transplanted cells and/or host cells
Ideal biomaterial :
biocompatible
promote cellular interaction and tissue development
possess proper mechanical and physical properties
32. Design and Selection of Biomaterials
Biocompatible
should be biodegradable and bioresorbable to support the
reconstruction of a completely normal tissue without
inflammation
degradation rate and the concentration of degradation
products in the tissues surrounding the implant must be at
a tolerable level
33. Design and Selection of Biomaterials
Promote cellular interaction and tissue development
should provide an appropriate regulation of cell behavior to
promote the development of functional new tissue
This is regulated by multiple interactions with the
microenvironment, including interactions with cell adhesion
ligands and with soluble growth factors
34. Design and Selection of Biomaterials
Promote cellular interaction and tissue development
provide temporary mechanical support sufficient to withstand in
vivo forces and maintain a potential space for tissue
development
The mechanical support should be maintained until the
engineered tissue has sufficient mechanical integrity to support
itself
35. Design and Selection of Biomaterials
Possess proper mechanical and physical properties
large ratio of surface area to volume is often desirable to allow the
delivery of a high density of cells
A high-porosity, interconnected pore structure with specific pore
sizes promotes tissue ingrowth from the surrounding host tissue
36. Types of Biomaterials
Three classes of biomaterials have been used
Naturally derived materials, such as collagen
Acellular tissue matrices, such as bladder and small-intestinal
submucosa
Synthetic polymers, such as polyglycolic acid (pga), polylactic acid
(pla), and poly(lactic-co-glycolic acid) (plga).
37. Types of Biomaterials
1) Collagen
Collagen is the most abundant and ubiquitous structural protein
in the body, and it may be
Readily purified with an enzyme treatment
Intermolecular cross-linking reduces the degradation by making
it less susceptible to an enzymatic attack
Contains cell-adhesion domain sequences that exhibit specific
cellular interactions
38. Types of Biomaterials
Can be processed into a wide variety of structures such as
sponges, fibers, and films
39. Types of Biomaterials
2)Alginate
Polysaccharide isolated from seaweed
Used as an injectable cell delivery vehicle and a cell
immobilization matrix
Copolymers of d-mannuronate and l-guluronate
Physical and mechanical properties are in proportion to the
length of poly-guluronate block
40. 3)Bioprinting
Natural materials such as have been used as “bio-inks” in
bioprinting technique based on inkjet technology
Materials can be “printed” into a desired scaffold shape
Living cells can also be printed using this technology
A three-dimensional construct containing a precise arrangement
of cells, growth factors, and ecm material can be printed
41. Types of Biomaterials
4)Acellular tissue matrices
collagen-rich matrices prepared by removing cellular
components from tissues
Slowly degrade after implantation and are replaced and
remodeled by ECM proteins synthesized and secreted by
transplanted or ingrowing cells
Mechanical properties of the acellular matrices are not
significantly different from those of native bladder submucosa
42. Types of Biomaterials
5)Synthetic polymers
Polyesters of α-hydroxy acids : PGA, PLA, and PLGA
Degradation products are nontoxic, natural metabolites that are
eventually eliminated from the body in the form of CO2 and
water
drawback of the synthetic polymers is lack of biologic
recognition. Needs incorporation of cell recognition domains
43. Types of Biomaterials
Thermoplastics: they can easily be formed into a three-
dimensional scaffold with a desired microstructure, gross shape,
and dimension by various techniques
44. Vascularization
A limiting factor for the engineering of tissues is that cells cannot
be implanted in volumes exceeding 3 mm3
Vascularization of the regenerating cells is essential to provide
nutrition and gas exchange beyond this maximal diffusion
distance
45. Vascularization
Two different processes:
Vasculogenesis, the in situ assembly of capillaries from
undifferentiated endothelial cells (ECs), and
Angiogenesis, the sprouting of capillaries from preexisting
blood vessels
46. Vascularization
Vasculogenesis
ECs are generated from precursor cells, called angioblasts,
ECs form the vessel primordia and aggregates, have no lumen;
a nascent endothelial tube is formed, composed of polarized
ECs;
a primary vascular network is formed from an array of nascent
endothelial tubes; and
pericytes and vascular smooth muscle cells are recruited
47. Vascularization
Angiogenesis
vasodilatation of the parental vessel, reducing the contact
between adjacent ECs;
degradation of the basement membrane
EC migration and proliferation to form a leading edge of the new
capillary;
generation of the capillary lumen and formation of a tube like
structure;
basement membrane synthesis; and
recruitment of pericytes and vascular smooth muscle cells
48. Vascularization
Three approaches
incorporation of angiogenic factors before implantation, to attract
host capillaries and to enhance neovascularization
seeding ECs with other cell types in before implantation eg.
smooth muscle cells and Ecs in penile corporeal tissue
Pre-vascularization of the matrix before cell seeding to form vascular
network, providing sufficient tissue perfusion