APPLICATIONS OF TISSUE

ENGINEERING

PRESENTED BY
ASMITA BENICHATAGE
MBT-09039
 A Definition

 Tissue engineering is an interdisciplinary field

that applies the principles of engineering and
the life sciences towards the development of
biological substitutes that restore, maintain,
or improve tissue function.
Langer and J. Vacanti “Tissue Engineering”. Science 1993.
 Engineering Strategies
 Engineering at the Genetic and Molecular Level
-Towards Genetically Designed Tissues for Regenerative Medicine
- Posttranscriptional Gene Silencing
- Biomolecule Use in Tissue Engineering

 Engineering at the Cellular Level

- Fetal Tissue Engineering: Regenerative Capacity of Fetal Stem Cells
- Embryonic Stem Cell Use
- The Unrestricted Somatic Stem Cell (USSC) From Cord Blood For Regenerative Medicine
- Mesenchymal Stem Cells: New Insights Into Tissue Engineering and Regenerative Medicine

 Engineering at the Tissue Level

- Cartilage Engineering
- Muscle Tissue Engineering
- Bone Tissue Engineering
- Tendon and Ligament Tissue Engineering: Restoring Tendon/Ligament and Its Interfaces
- Neural Tissue Engineering and Regenerative Medicine

 Engineering at the Organ Level

-Breast Tissue Engineering
-Bioartificial Liver
-Pancreas Engineering
-Tissue-Engineered Urinary Bladder
-Cell-Based Regenerative Medicine for Heart Disease
APPLICATIONS
 In Neurology
 In Maxillofacial Surgery
 In Dental Implantology
 In General Surgery
 Regeneration of Renal Tissues
 In Plastic Surgery
 Cardiovascular Substitutes
 Skin Substitutes
 In Orthopedic Surgery
 In Endocrinology
 In Hematology
How to make or modify tissues
 In vitro tissue  In vivo tissue
engineering engineering

 Ex vivo tissue
engineering

Langer and J. Vacanti “Tissue

Engineering”. Science, 1993.
 General Steps For Tissue Engineering

(1) Start building material (e.g., extracellular matrix,

biodegradable polymer)
(2) Shape it as needed
(3) Seed it with living cells
(4) Bathe it with growth factors
(5) Cells multiply & fill up the scaffold & grow into
three-dimensional tissue
(6) Implanted in the body
(7) Cells recreate their intended tissue functions
(8) Blood vessels attach themselves to the new tissue
(9) The scaffold dissolves
(10) The newly grown tissue eventually blends in with its
surroundings
Tissue Engineering Of Cultured Skin
Substitutes
Introduction

 Our skin is a major organ of the body that acts as

a barrier to pathogens and trauma – Largest
laminar organ.
 Skin defects caused by burns, venous and
diabetic ulcers, or acute injury occasionally
induce life-threatening situations.
 Thus, the need for a functional and cost-effective
permanent skin substitute for burn victims has
always been garnered.
The Anatomy of Human Skin

 Epidermis (5 layers)
Keratinocytes provide protective properties.
Melanocytes provide pigmentation.
Langerhans’ cells help immune system.
Merkel cells provide sensory receptors.
 Dermis (2 layers)
Collagen, glycoaminoglycans, elastine, ect.
Fibroblasts are principal cellular constituent.
Vascular structures, nerves, skin appendages.
 Hypodermis (fatty layer)
Adipose tissue plus connective tissue.
Anchors skin to underlying tissues.
Shook absorber and insulator.
 General Design Properties

 Essential Design Properties

 "The initial replacement material should provide immediate
physiologic wound closure and be eliminated once it has provided
sufficient information for reconstitution of neodermis”
 It should protect the wound by providing a barrier to the outside
 It should control water evaporation and protein and electrolyte loss
 It should limit excessive heat loss
 It should decrease pain and allow early mobilization
 It should provide an environment for accelerated wound healing
 The risk of infection must be taken into account
Currently Available Skin Substitutes
According To Their Fundamental Features
 Skin substitutes classified as
 Permanent or temporary
 Epidermal, dermal or composite
 Biological or alloplastic (synthetic)

 Temporary-
material designed to be placed on a fresh wound (partial thickness) and left
until healed

 Semi-permanent-
material remaining attached to the excised wound and eventually replaced by
autogenous skin grafts

 Permanent-
incorporation of an epidermal analogue, dermal analogue, or both as a
permanent replacement
 Cont.
 Biologicals (Naturally occurring tissues)
– Cutaneous allografts
– Cutaneous xenografts
– Amniotic membranes

 Skin substitutes
– Synthetic bilaminates
– Collagen-based composites

 Collagen-based dermal analogues

– Deepithelized allografts

 Culture-derived tissue substitues

– Cultured autologous keratinocytes (sheet grafts, cell suspensions)
– Bilayer human tissue
– Polyglycolic or acid mesh
– Fibroblast-seeded dermal analogs
– Collagen-glycosaminoglycan matrix
– Epithelial seeded dermal analogs
Problems of Skin Substitution in Major
Burns
 Rapid and effective burn wound closure-imp aspect

 Early surgical removal of heat-denatured proteins

and devitalized tissue from a wound

 Lack of autograft- overcome by use of synthetic

replacements

 Initial use of allografts may undergo immunogenic

rejection
 Cultured Skin Developments
Rheinwald JG, Green H.
Serial cultivation of strains of human epidermal keratinocytes
Human diploid epidermis epidermal cells were successfully grown in serial culture.
To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts
must be controlled so that the epidermal cell population is not overgrown.
Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density.
When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human
fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies.
Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which
the dividing cells are confined to the lowest layer(s).
Hydrocortisone is added to the medium, to make the colony morphology more oderly and
distinctive, and maintains proliferation at a slightly greater rate.
Under given culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts.
Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime.
The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
Epidermis Anatomy- Here are four major layers of keratinocytes (the
structural cells) in the epidermis.
The stratum basale, has cells that are shaped like columns.
In this layer the cells divide and push already formed cells into
higher layers.
As cells move into the higher layers, they flatten and eventually die.
 Cont.
 This technique allows growth of the epidermis from a single small skin
biopsy within 3–4 weeks

 2° culture of isolated colonies of epithelial cells

 Approaches for skin culture substitutes in vitro

 Cell culture of multilayered epithelial transplants (commonly termed “sheet

grafts”)

 Growth and construction of composite multilayered dermal-epidermal

analogues

 Growth and transplantation of pre-confluent cell grafts (cultured or non-

cultured)
 Cultured “Sheet Grafts” (Cultured
Epithelial Autografts- CEA)
 The growth of multilayered cultured epithelial
sheets of human autologous keratinocytes-
Permanent substitute
 Affecting factors
 High cost
 Hospital readmissions for reconstruction of
contractures
 Wound infection- fragile epithelium and partially
developed dermoepidermal junction
 Thus 2° devices necessary to manipulate CEA to
handle fragile cell layers
 Less adherence to wound region
Cultured Human Cell Suspension
Grafting
 Experimentally, epidermal cell suspensions were transplanted
in saline solutions to full-thickness pig wounds, in 1952-no
consistent result
 Hunyadi reported successful transplantation of non-cultured
keratinocytes, gained by trypsinization from biopsies and
suspended in a fibrin matrix to heal chronic venous leg ulcers
 Suspending the cells in a fibrin sealant versus saline solution
 Fibrin is a naturally occurring substrate - key role in all wound
healing
 Fibrin-cultured autografts - applied on massive full-thickness
burns,
 It allows reduction in cost , problems related to handling
Cont.
 Such keratinocyte-fibrin suspensions(KFSs) can be
made available for grafting within 10 days of
seeding
 Thus KFSs preferred over CEA (3-4 weeks)
 Lack of dermal structures using keratinocyte alone
lead to combination of
 a preliminary wound bed preparation by allografts,
 followed by subsequent KFS transplantation
together with meshed split-thickness allograft skin
as an overlay
Alloplastic or Mixed Synthetic-
Biological Cell Carriers
 Problems with purely epithelial cell grafts have induced the combination of
cultured autologous keratinocytes with various alloplastic dermal
regeneration templates
 trypsinized whole-skin-cell suspensions (uncultured keratinocytes and
fibroblasts) centrifuged into a collagen-glycosaminoglycan (C-GAG) matrix
and grafted onto guinea pigs were able to facilitate the regeneration of a
healthy epidermis
 Compound materials has been propagated for reconstructive and burn
surgery that consists of
(1) a well-characterized, so-called “dermal portion” that consists of a porous
lattice of fibres of a cross-linked bovine collagen and GAG that is supposed
to be replaced by new collagen synthesized by fibroblasts that grow in on
fullthickness wounds
(2) a so-called “epidermal” cover of synthetic polysiloxane polymer (silicone)
 Collagen integrates into wound bed, silicone layer peeled off
Cultured Cells and Biological Carriers

 Pre-confluent human keratinocyte monolayer grafts

have also been used in combination with biological
carrier materials
 proliferating basal cells which are responsible for the
initial reformation of an epithelium can be
transplanted upside down with the carrier on top as a
bio-dressing
 One of the most common materials has been C-GAG,
with or without the cover of a gas-permeable silastic
membrane that serves as a barrier to fluid loss
 Many unsolved mysteries yet to solve…
 Research aim is to replace with a truly functional
skin inclusive of all dermal appendages that are
of permanent skin-like quality
 perhaps the definitive breakthrough will come
through the ability to replace worn out,
defective or damaged body parts using
technologies that resemble natural
regeneration.
REFERENCES

 “Fundamentals of Tissue Engineering and

Regenerative Medicine”, by U. Meyer et al
 “Essentials of Stem Cell Biology”, by Robert
Lanza
 Rheinwald JG, Green H. Serial cultivation of
strains of human epidermal keratinocytes: the
formation of keratinizing colonies from single
cells. Cell 6 (3): 331–43, 1975
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