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.

1. Principles
of
Tissue Engineering
Dr Roshan V Shetty
Senior Resident In Urology
AJ IMS & RC

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.

29. Summary
Stem Cells
Reproductive/Therapeutic
Cloning (SCNT)
Reprogramming
(iPSC)
Native Targeted
Progenitor
Cells
Embryonic stem cells
Perinatal Stem cells
Adult stem cells

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

49. Stem cells Biomaterials Vascularisation
Engineered Tissue for Regeneration

50. Thank You !