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Tissue Engineering of Skin
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Tissue Engineering of Skin
CHE 554: Principles of
Tissue Engineering
11/10/08
Aditya Shah
Karthik
Gopalakrishnan
Tissue Engineering of Skin
Overview
• Introduction
• Anatomy of the skin
• Role of Tissue Engineering
• Applications
• Commercial Aspects
• Challenges
• Future
• Conclusion
• References
Tissue Engineering of Skin
Introduction
Tissue Engineering of Skin
• The aim of skin engineering is to create a functional
biological substitute to restore homeostasis of the body
• Use of fundamentals of medicine, biology, engineering
and biotechnology
• Approximately 500,000 surgical procedures performed
every year in the U.S.
Tissue Engineering of Skin
• Gold standard approach was widely used before the
introduction of the Tissue Engineered Skin
• Currently available skin substitutes, include: autografts,
allografts, xenograft and synthetic materials that are
being implanted.
• The use of grafts is very important
Tissue Engineering of Skin
Anatomy of The Skin
Tissue Engineering of Skin
Tissue Engineering of Skin
• Skin- Largest organ on the human body.
• Accounts for 15% of the body weight and is more than the
weight of the single internal organ of the body.
• A special field of study Dermatology is used to study the
nature of the skin and its behavior.
Tissue Engineering of Skin
Skin is composed of three layers
1. Epidermis: The outermost layer
2. Dermis: The middle layer
3. Hypodermis: The lowermost layer
Tissue Engineering of Skin
Epidermis• The outermost layer
• Forms the protective wrap over the body’s surface.
• Does not have blood vessels
• Nourished by diffusion from the dermis
• Epidermis is subdivided into 5 layers:
1. Stratum corneum
2. Stratum lucidum
3. Stratum granulosum
4. Stratum spinosum
5. Stratum germinativum
Tissue Engineering of Skin
• The main type of cells are
keratinocytes,melanocytes,langerhans cells and
Merkel cells.
• Melanocytes- Produces melanin which gives skin its
color and protects from the harmful UV radiation
• keratinocytes, Produces keratin which is a water
repellent body which gives the epidermis its strength.
• Merkels- Large oval cells which give the skin its
touch.
Tissue Engineering of Skin
Dermis• Varies in thickness according to location
• Three types of tissues:
1. Collagen
2. Elastic tissue
3. Reticular fibers
• Two layers of Dermis:
Papillary : has thin arrangements of collagen fibers.
Reticular layers: thicker than papillary layer.
Tissue Engineering of Skin
Specialized cells of Dermis has:
• Hair follicles
• Sebaceous (oil) glands and apocrine (scent) glands
• Also contains eccrine (sweat) glands but are not
associated with hair follicles.
• Blood vessels and nerves has a network through this
layer.
• The nerve transmits sensations of touch, pressure, pain,
itch and temperature.
Tissue Engineering of Skin
Hypodermis
• Has layer of fat and connective tissue that houses larger
blood vessels and nerves.
• The size of this layer varies throughout the body and from
person to person.
• Important in the regulation of temperature of the skin
itself and the body.
Tissue Engineering of Skin
Functions of skin
• To maintain homeo-stasis of the body.
• To provide protection, sensation, absorption, storage and
synthesis, excretion, heat regulation, control evaporation
of body fluids.
Tissue Engineering of Skin
Role of Tissue Engineering
Tissue Engineering of Skin
Why do we need Engineered Skin to progress?
• Promote wound healing
• Moderate the pain due to wound
• Higher rate of cell proliferation in in vitro culture
• Optimum use of biomaterials and field allied to it
• Technology available to patients easily
• Better quality of life for patient
• Affordable
Tissue Engineering of Skin
Main Principles of Tissue Engineering
• The cell source is identified, separated and also produced in ample numbers
• The alternative biocompatible material which could be used as a cell substrate or cell encapsulation material isolated or synthesized
• The cells seeded or onto the material must maintain the exact function, morphology
• The engineered structure is then placed into the appropriate in vivo site
Tissue Engineering of Skin
Process
• Start building material• Shape the material• Seed it with living cells • Bathe it with growth factors• Cells multiply & fill up the scaffold & grow into three-
dimensional tissue• Implanted in the body• Cells recreate their intended tissue functions• Blood vessels attach themselves to the new tissue• The scaffold dissolves• The newly grown tissue eventually blends in with its
surroundings
Tissue Engineering of Skin
Scaffolds
Properties of Scaffolds
• Structural Integrity
• Exact pore size
• Mechanical strength
• Biodegradable
Tissue Engineering of Skin
Synthesis of Tissue Engineered Scaffolds
Methods
• Nanofiber Self-Assembly
• Textile technologies
• Solvent Casting & Particulate Leaching (SCPL)
• Gas Foaming
• Emulsification/Freeze-drying
• Thermally Induced Phase Separation (TIPS)
Tissue Engineering of Skin
Materials used for making Scaffolds
Natural
• Collagen
• Fibronectin
• Gelatin
Synthetic
• PEGTpoly(ethyleneglycol terephthalate)/PBTpoly(butylene
terehthalate)
• PLGA poly(Dl-lactide-co-glycolide)
Bio Synthetic
• Collagen with amniotic membrane component
• Dithio-bis-propionimidate (DTBP) crosslinked Chitosan
Tissue Engineering of Skin
Collagen
• Synthesized by Fibroblasts and Myofibroblasts
• Present in Dermis
Tissue Engineering of Skin
Types of Collagen
• Type I Collagen - The most abundant collagen in the body.
Found in tendons, bones, skin and other tissues.
Particularly abundant in the scar tissue.
• Types II, IX, X, XI - Cartilage
• Type III - Common in fast growing tissue, particularly at the
early stages (Phase 1) of wound repair. Much of it is
replaced later by the type stronger and tougher type I
collagen.
Tissue Engineering of Skin
• Type IV - Basal lamina (filtration membrane of capillaries)
• Type V, VI - Generally found alongside type I
• Type VII - Epithelia (lining of GI tract, urinary tract, etc.)
• Type VIII - Lining of blood vessels
• Type XII - Found alongside and interacts with types I and
III
Tissue Engineering of Skin
Fibronectin
• Fibronectin is major multifunctional component of the ECM
• While Designing a fully functional skin the major consideration is about the mechanical forces and interactions brought about by wound contraction
• Wound contraction involves a complex interplay between the fibroblast cytoskeleton and integrins with their ECM ligands
• Mechanical stretching affects fibronectin function
• Fibrin, associated with fibronectin, has been shown to support keratinocyte and fibroblast growth both in vitro and in vivo, and enhance cellular motility in wounds
Tissue Engineering of Skin
PEGT/PBT/PLGA
• Non Toxic
• Biodegradation could be increased
Tissue Engineering of Skin
DTBP Crosslinked CHITOSAN
•Chitosan is deacetylated
derivative of chitin
•Improved tensile strength with
80 & 90% chitosan
•Improved porosity with
crosslinked DTBP
•Reduced and improved
biodegradation rate
Tissue Engineering of Skin
Applications
Tissue Engineering of Skin
Areas where Tissue Engineering of Skin can be used
1. Diabetic sores and ulcers
2. Healing and replacement of burned skin
3. Reduction in scar formation
4. Plastic/ Cosmetic Surgery
Tissue Engineering of Skin
Diabetic sores and ulcers
•Diabetic sores accounts for
almost 50% of total patients
admitted to hospitals for diabetic
related problems
•Nerve damage can lead to
numbness of the infected part
Tissue Engineering of Skin
•Damaged to nerves leads to
less oxygen and blood
circulation in the affected area
•Injuries commonly lead to
infection or gangrene
•High infection may lead to
amputation of the infected part
Tissue Engineering of Skin
Treatments for Diabetic Ulcers
• Antibiotics- Nitric oxide is being investigated as it is a
powerful vasolidator
• Antibiotics can be used to reduce the putrid smell of
infection
• Use of light therapy
Tissue Engineering of Skin
•Advanced dressing techniques
using synthetic Dressing,
biological dressing, bio-synthetic
dressing or cultured keratinocyte
dressing
Tissue Engineering of Skin
Burns
•Approximately 2,150,000
patients are treated in the U.S
alone for burn related
complications
•Seriousness of a burn injury
depends on how deep the injury is
and how much of the body has
been burned.
Tissue Engineering of Skin
Degrees of Burns
•First degree burns affect the
outer layer of the skin, causing
pain, redness, and swelling.
•Injury to the second layer of skin
(the dermis) is called second
degree burns.
•Third-degree burns extend into
deeper tissues, causing brown or
blackened skin that may be
numb.
Tissue Engineering of Skin
Treatment for Burns
• Two major types of Surgical Procedures that can help to
conceal scarring and replace lost tissue for severe burn
victims:
1. Dermabrasion.
2. Skin Grafts.
Tissue Engineering of Skin
•Dermabrasion is a surgical
procedure to improve or
minimize the appearance of
scars, restore function and
correct disfigurement resulting
from an injury.
Tissue Engineering of Skin
•A Skin Graft is surgical
procedure in which a piece of
skin from one area of the
patient's body is transplanted to
another area of the body.
•Types of grafts available:
Pinch grafts
Split-thickness grafts
Full thickness grafts
Tissue Engineering of Skin
Scars
Three types of scars
• Keloid
• Hypertrophic
• Contractures
Tissue Engineering of Skin
Keloid scars
• Scars will grow beyond the site
of injury
•They occur when the body
continues to produce collagen a
tough fibrous protein, after the
wound has healed.
•Cell type termed fibrocyte with
a distinct phenotype originally
described as
collagen+/vimentin+/CD34+.
Tissue Engineering of Skin
•CD34+ expression decreases
with time whilst expression of
proline-4-hydroxylase increases ,
an enzyme required for
producing collagen
•Research on sub-lethally
irradiated mice shows that the
reversion of CD34 expression
may represent a decrease in
collagen synthesis
An incisional back wound at day 10 post-wounding in a C57BL/6 mouse. Sub-lethally irradiated C57BL/6 mice underwent bone marrow reconstitution with bone marrow from C57BL/6 mice
Tissue Engineering of Skin
Hypertrophic Scars
•Hypertrophic scars are red, thick
and raised.
•Do not extend beyond the site of
injury or incision.
•Hypertrophic scars have
oriented collagen
•Gets better with time, however
can be reduced by use of steroids
and injections
Tissue Engineering of Skin
Contractures
•Occurs mainly due to burn
injuries
• It is a permanent tightening of
skin .
•Makes the tissues resistant to
stretching and prevents normal
movement of the affected area.
Tissue Engineering of Skin
Treatment for Scars
Inhibiting overproduction of collagen and reduction of
fibrosis.
Treatment includes:
• Surgical correction
• Compression therapy
• Irradiation
Tissue Engineering of Skin
Plastic/cosmetic Surgery
•Interested in the correction of form
and function.
•Famous for aesthetic surgery.
• Plastic surgery also includes a variety
of fields:
-craniofacial surgery
-burn surgery
-microsurgery
-pediatric surgery
-congenital defect corrections
Tissue Engineering of Skin
•Skin tissue (skin grafting) is one of
the most common procedures.
•Liposuction, breast augmentation,
eyelids surgery, face lift, tummy
tuck, collagen injections, chemical
peel, laser skin resurfacing,
rhinoplasty, burns, traumatic
injuries such as facial bone
fractures, removal of cancers or
tumors, scar reduction
Tissue Engineering of Skin
Commercial Aspects
Tissue Engineering of Skin
INTEGRA®
•Designed to manage major burns.
•Has a pore volume fraction of 98%. M.W. of
crosslinks are in the range of 12K+5K dalton
with an average pore diameter of 30-
120µm.
•Comprises of bovine collagen and shark
chondroitin sulphate with silicone
membrane that acts as a temporary barrier.
Tissue Engineering of Skin
• The material is grafted onto the wound bed and under
this membrane vasculogenesis occurs. Once the
vascularization is complete silicon membrane barrier is
replaced with patients own cells.
Tissue Engineering of Skin
INTEGRA® Procedure in brief
• Week1:
-Integra is applied to the burn injury or scar contracture and is assessed then
evaluated.
-Wound excised surface fluids evading help it to attach .
-The wound is cleaned and the damaged tissue or contracture scar is
completely excised
down to viable tissue in preparation for the application of the INTEGRA
template.
• Week 2: Dermal cells start migrating in matrix and collagen in INTEGRA is
replaced by organic
collagen produced by new dermal cells
• Week 3: Silicone removal and applying epidermal autograft.
• Week 4-8: Successful engraftment of skin
Tissue Engineering of Skin
Problems with INTEGRA®
• Bovine collagen may bring forth the antigenic response.
• They fail to adhere securely.
• Three weeks are required to expand the dermal autograft.
Tissue Engineering of Skin
Dermagraft®
• A hope for faster recovery for Diabetic foot ulcers (DFU)
• Dermagraft® is a cryopreserved human fibroblast-derived
dermal substitute; it is composed of fibroblasts derived
from newborn foreskin tissue, extra cellular matrix, and a
bioabsorbable scaffold.
• Neonatal fibroblasts rapidly proliferate to produce
collagen,GAGs and growth factors to induce wound healing
Tissue Engineering of Skin
ADVANTAGES
•Used in treatment of DFU which if
left untreated may lead amputation
of foot.
•Dermagraft increases blood flow to
DFU which results in faster healing
•Only disadvantage is sub
membrane fluid accumulation is
more in Dermagraft.
Tissue Engineering of Skin
ICX-SKN by INTERCYTEX
•Aimed to provide better healing for
burns and acute wounds victims.
•Comprises of allogeneic human dermal
fibroblasts set in a natural human
collagen matrix, which mimics the
structure of skin and is intended as a
skin graft replacement. An additional
layer of human keratinocytes may be
included to form an epidermal layer.
•Completed Phase I trials. Phase II
expected to begin in 2009
Tissue Engineering of Skin
• Other Skin graft substitutes available in the market are
Transcyte®, Apligraft®, Orcel®, Biobrane.
• Cell delivery approaches are the next upcoming field in
Tissue
Engineering of Skin.
• Keratinocytes can be grown into integrated cell sheets,
made into suspensions and sprayed onto wound sites with
or without fibrins. Eg.: CellSpray®
Tissue Engineering of Skin
• Myskin® is another product which is based on same
principle except chemically defined polymer carrier
dressing.
• Epidex® uses cells cultured from hair follicles and
delivered as small sheets
Tissue Engineering of Skin
Challenges
Tissue Engineering of Skin
• Using the right kind of serum: The widely used culture
methodology uses murine fibroblasts and bovine serum.
Murine cells might contain viruses able to transform
human cells.Bovine serum may be infected by BSE.
• Biocompatibility, mechanical & handling properties.
• Has many clinical benefits but many expectations both
clinical as well as commercial are unrealistic.
Tissue Engineering of Skin
• Treating deeper wounds.
• Better adhering of keratinocytes to the dermis site.
• Cost of automation too high for the companies to survive.
• Lots of regulation laid by FDA .
Tissue Engineering of Skin
Future
Tissue Engineering of Skin
• Human embryonic stem cells (hESCs) are a promising
source of skin cells for engineered skin equivalents as
they have a potential to be a safe source of unlimited
amount of skin cells.
• Better adhesion of keratinocytes using gene therapy.
Tissue Engineering of Skin
• Nanofibers, which mimic collagen fibrils in the
extracellular matrix (ECM), can be created from a host of
natural and synthetic compounds and have multiple
properties that may be beneficial to burn wound care.
• Cost efficient.
• Less stringent regulations.
Tissue Engineering of Skin
Conclusion
Tissue Engineering of Skin
• Many important clinical milestones have been reached and the
number of artificial skin substitutes licensed for clinical use is growing,
but they have yet to replace the current ‘‘gold standard’’ of an
autologous skin graft.
• Currently available skin substitutes often suffer from a range of
problems that include poor integration which in many cases is a direct
result of inadequate vascularisation, scarring at the graft margins and
a complete lack of differentiated structures.
• The ultimate goal for skin tissue engineers is to regenerate skin such
that the complete structural and functional properties of the wounded
area are
restored to the levels before injury.
• New synthetic biomaterials are constantly being developed that may
enable control over wound repair and regeneration mechanisms by
manipulating cell adhesion, growth and differentiation and
biomechanics for optimal tissue development.
Tissue Engineering of Skin
References
Tissue Engineering of Skin
• http://ithaca.rice.edu/kz/USAToday/SkinArticle.htm
• http://www.cbte.group.shef.ac.uk/research/te2.html
• http://ouroboros.wordpress.com/2007/02/21/tissue-engineering-the-
skin/
• http://findarticles.com/p/articles/mi_qa3977/is_200005/ai_n8902669/
pg_2?tag=artBody;col1
• http://en.wikipedia.org/wiki/Tissue_engineering
• Progress and opportunities for tissue-engineered skin, S. MacNeil,
Nature, 445, 874-880 (2007)
• Tissue engineering of replacement skin: the crossroads of
biomaterials, wound healing, embryonic development, stem cells and
regeneration, A. D. Metcalfe and M. W. Ferguson, J. R. Soc. Interface,
4, 413-437 (2007)
Tissue Engineering of Skin
• Emerging Treatments in Diabetic Wound Care by Thanh Dinh,
DPM;1 Hau Pham, DPM;1 Aristidis Veves, MD2-VOLUME: 14
PUBLICATION DATE: Feb 10 2002 Issue 1
• Skin substitutes from cultured cells and collagen-GAG polymers-
S. T. Boyce
• http://www.intercytex.com/icx/products/woundcare/icxskn/
• http://www.burncarerehab.com/pt/re/jburncr/abstract.01253092-
2008090
• http://www.burnsurvivor.com/surgical_procedure_skingrafts.html
• Picture source: http://www.images.google.com
• Video source: http://www.ilstraining.com/idrt/videos.html
Tissue Engineering of Skin
• In vitro skin irritation: facts and future. State ofthe art review of
mechanisms and models. Toxicol. In Vitro 18, 231–243 (2004).by
Welss, T., Basketter, D. A. & Schroder, K. R.
• Tissue Engineering: Palsson, Bernhard & Bhatia, Sangeeta ;
Pearson Prentice Hall Bioengineering.
• Methods of Tissue Engineering: Atala, Anthony & Lanza Robert;
Academic Press.
Tissue Engineering of Skin
Questions?
Tissue Engineering of Skin
Thank You!