Presentation

Tissue Engineering of Skin

CHE 554: Principles of

Tissue Engineering

11/10/08

Aditya Shah

Karthik

Gopalakrishnan


Overview

• Introduction

• Anatomy of the skin

• Role of Tissue Engineering

• Applications

• Commercial Aspects

• Challenges

• Future

• Conclusion

• References


Introduction


• 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.


• 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


Anatomy of The 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.


Skin is composed of three layers

1. Epidermis: The outermost layer

2. Dermis: The middle layer

3. Hypodermis: The lowermost layer


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


• 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.


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.


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.


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.


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.


Role of Tissue Engineering


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


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


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


Scaffolds

Properties of Scaffolds

• Structural Integrity

• Exact pore size

• Mechanical strength

• Biodegradable


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)


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


Collagen

• Synthesized by Fibroblasts and Myofibroblasts

• Present in Dermis


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.


• 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


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


PEGT/PBT/PLGA

• Non Toxic

• Biodegradation could be increased


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


Applications


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


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


•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


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


•Advanced dressing techniques

using synthetic Dressing,

biological dressing, bio-synthetic

dressing or cultured keratinocyte

dressing


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.


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.


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.


•Dermabrasion is a surgical

procedure to improve or

minimize the appearance of

scars, restore function and

correct disfigurement resulting

from an injury.


•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


Scars

Three types of scars

• Keloid

• Hypertrophic

• Contractures


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+.


•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


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


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.


Treatment for Scars

Inhibiting overproduction of collagen and reduction of

fibrosis.

Treatment includes:

• Surgical correction

• Compression therapy

• Irradiation


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


•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


Commercial Aspects


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.


• 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.


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


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.


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


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.


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


• 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®


• 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


Challenges


• 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.


• 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 .


Future


• 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.


• 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.


Conclusion


• 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.


References


• 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)


• 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


• 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.


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