The use of Stem cells in burn care

Yefta Moenadjat

Introduction

Burn• Tissue disruption due contact to source: thermal

(burn/scald), chemical, electrical (lightning), radiation.• Problems encountered:

– Acute phase• Deterioration of airway, breathing and circulation• Systemic Inflammatory Response Syndrome

(SIRS) & Multi-system Organ Dysfunction Syndrome (MODS)

– Late phase• Wound healing

Introduction

Burn• Tissue disruption: wound classification

Type of wound1 Superficial burn 1o

2 Partial thickness burn 2o

• superficial partial thickness

2o superf• deep partial thickness(full thickness) 2o deep

3 Deep burn 3o

Superficial burn

• Epidermal layer disruption• Intact dermal • Painful • Spontaneous healing 5-7 days• Treatment:

• Moisturizer cream• Common analgesic

Partial thickness burn

1. Superficial

• Epidermal layer disruption• 1/3 superficial dermal layer• Blister formation• Painful • Intact dermis >Spontaneous healing 10-14

days• Treatment:

• Moisturizer cream• Blister management• Common analgesic

Partial thickness burn

2. Deep (full thickness)

• Epidermal layer disruption• 2/3 superficial dermal layer• Blister formation (+/─)• Thin eschar• Painful • Intact skin appendicesSpontaneous healing up to 21

days• Treatment:

• Moist dressing• Blister management• Common analgesic

Deep burn

• Epidermal layer, dermal layer and deeper layer (subcutaneous & adipose tissue, muscles, bones)

• Eschar• No sensation • Intact dermis & skin appendices

(─)Spontaneous healing impossible

• Treatment:• Debridement (escharectomy)• Skin grafting

Surgical Non surgicalConcept changes:

• Total excision• Tangential excision• Total excision

Second place

Rapid Slow Complication:Surgical bleeding

No bleeding complication

Method:• Conventional (Humby

knife)• Electric dermatome• Hydropressure

Method:• Autolytic • Enzymatic

Débridement:

Debris removal referred to source control

The wound closure

• Problems encountered in burn wound closure

• Degree of severity– Damaged tissue → deteriorated circulation→ non

vital tissue → inflammatory response ▲– Burn exhaustion [metabolic changes,

inflammation] – Prolonged phases of wound healing

• Impaired – non healing wound

Wound healing: Review

0 2 4 6 8 365

Injury Days after injury

Homeostasis &inflammation

FibroplasiaMaturation

Phases of Wound healing

• Template formation– Proliferation of fibrin cells [collagen matrix ] replaces the

clot• Angiogenesis

– Proliferation of endothelial cells [new vessels formation]

• Epithelialization– Proliferation of epithelial cells from wound edges and

skin appendices

Phase of proliferative [fibroplasias, fibro-proliferative]

►Granulation tissue

Phase of Fibroplasias

1. Fibrin proliferationearly

Phase of Fibroplasias

1. Fibrin proliferationearly

1. Fibrin proliferationearly

Phase of Fibroplasias Phase of Fibroplasias

1. Fibrin proliferationlate

Phase of Fibroplasias

1. Fibrin proliferationlate

Phase of Fibroplasias

2. Angiogenesisearly

Phase of Fibroplasias

2. Angiogenesisearly

Phase of Fibroplasias

2. Angiogenesislate

Phase of Fibroplasias

Granulation: Fibrin proliferation + Angiogenesis

Phase of Fibroplasias

Granulation: Fibrin proliferation + Angiogenesis

Wound: contraction

Phase of Fibroplasias

3. Epithelializationearly

Phase of Fibroplasias

Phase of Fibroplasias

3. Epithelializationcomplete

Epithelialization:Epithelization is start from the wound edges

Basal membrane

Epithelialization:Epithelization is not always start from the wound edges

Hair follicleSebaseous gland

Sweat gland

• Collagen deposition [early, 2 mo]– Indurative tissue

• Collagen resorption [late, up to 8-12 mo]– Soften tissue

• Regression of vessels– Tissue becomes pale

Phase of remodeling [phase of maturation]

►Scar tissue

Conditions of fibroplasias

• Healthy granulation tissues– Adequate collagen

matrix– Angiogenesis

Granulation tissue

►Firm and pale tissue

Conditions of fibroplasias

• Healthy granulation tissues– Adequate collagen

matrix– Angiogenesis

Granulation tissue

►excessive granulation tissue

Influencing factors

•• Structural component or scaffolding Structural component or scaffolding •• Biologically active component stimulating all phases of Biologically active component stimulating all phases of

healing healing •• Collagen (protein) Collagen (protein)

–– Scaffold for cell migration and matrix depositionScaffold for cell migration and matrix deposition–– Cell guidanceCell guidance

•• Elastin (protein) Elastin (protein) –– Tissue elasticityTissue elasticity

•• Fibronectin (protein) Fibronectin (protein) –– Cell to cell adherenceCell to cell adherence–– Contact orientation for cellsContact orientation for cells–– Increases epithelial cell division, migrationIncreases epithelial cell division, migration–– Chemo attractant for fibroblasts, macrophagesChemo attractant for fibroblasts, macrophages

Influencing factors

•• Growth Factors (proteins) Growth Factors (proteins) •• Stimulate all phases of wound healingStimulate all phases of wound healing•• Glycosaminoglycan (glycosylated protein) Glycosaminoglycan (glycosylated protein)

–– Cell adherence propertiesCell adherence properties–– Conduit for healing factorsConduit for healing factors–– Deactivator of proteasesDeactivator of proteases–– Scaffold or foundation for dermal elementsScaffold or foundation for dermal elements

•• Hyaluronic Acid (complex carbohydrate) Hyaluronic Acid (complex carbohydrate) –– Maintaining matrix hydratedMaintaining matrix hydrated–– Decreases inflammationDecreases inflammation–– Stimulates healingStimulates healing–– Proper cell alignmentProper cell alignment

•• InjuryInjury•• InflammationInflammation•• Inadequate blood flowInadequate blood flow•• IschemiaIschemia--reperfusion injuryreperfusion injury•• InfectionInfection

SIRS and MODS

Toxins AutolysisInflammation Infection Healing

↑ demand forInflammatory cells

↑ demand forImmune modulation

↑ demand forMesenchymal stem cells

Marrow Exhaustion

↑ demand onBone Marrow supportMarrow suppression

Product Company Tissue of Origin Layers Category Uses How

supplied

Human allograft Skin bank Human

cadaverEpidermis and dermis

Split thickness skin

Temporary coverage of large excised burns

Frozen in rolls of varying size

Pig skin Xenograft

Brennan Medical St. Louis, Mo

Pig dermis Dermis Dermis

Temporary coverage of partial thickness and excised burns

Frozen or refrigerated in rolls

Human amnion

On site procurement Placenta Amniotic

membrane

Epidermis Dermis Same as

aboveRefrigerator

Oasis® Healthpoint LTD San Antonio, Tx

XenograftExtracellular wound matrix from small intestine submucosa

Bioactive Dermal like Matrix

Superficial burns Skin graft donor sites Chronic wounds

Room T°storage Multiple sizes 3x3.5cm 7x20cm

Temporary Skin Substitute

Product Company Tissue of Origin Layers Category Uses How

supplied

Biobrane® Dow Hickam/Bertek Pharmaceuticals

Synthetic with added denatured bovine collagen

Bilayer product outer silicone Inner nylon mesh with added collagen

Synthetic epidermis and dermis

Superficial partial thickness burns,Temporary cover of excised burns

Room T°storage 15x20inch 10x15cm 5x15inch 5x5 inch

Transcyte® Smith & Nephew Wound Management Largo, FL

Allogenic Dermis

Bilayer product Outer silicone Inner nylon seeded with neonatal fibroblasts

Bioactive Dermal Matrix Components on Synthetic dermis and epidermis

Superficial to mid-Partial thickness burns Temporary coverage of excised burns

Frozen in 5x7.5 inch sheets

Temporary Skin Substitute

AVAILABLE PERMANENT SKIN SUBSTITUTES

Product Company Tissue of Origin Layers Category Uses How

supplied

Apligraf

Organogenesis Inc and Novartis Pharmaceuticals

Corp

Allogenic Composite

Collagen matrix seeded with

human neonatal keratinocytes and fibroblasts

Composite:

Epidermis and

Dermis

Chronic wounds,

often used with thin

STSG Excised

deep burn

7.5cm diameter

disc 1/pack

OrCel Ortec International

Inc.

Allogenic Composite

Collagen sponge seeded with

human neonatal keratinocytes and fibroblasts

Composite:

Epidermis and

Dermis

Skin graft donor site,

chronic wounds

6x6cm sheets

Epicel* Genzyme Tissue Repair Corp

Autogenous keratinocytes

Cultured autologous

keratinocytes

Epidermis Only

Deep partial and

full thickness

burns >30% TBSA

50cm2

sheets in culture medium

1. Used mainly in burns

Permanent Skin Substitute

AVAILABLE PERMANENT SKIN SUBSTITUTES

Product Company Tissue of Origin Layers Category Uses How

supplied

Alloderm Life Cell Allogenic dermis

A cellular Dermis (processed allograft)

Dermis only

Deep partial and full thickness burns, Soft tissue replacement, Tissue patches

1x2cm to 4x12cm

Integra* Integra Life Science Corp Synthetic

Silicone outer layer on collagen GAG dermal matrix

Biosynthetic Dermis

Full thickness soft tissue defects definitive “closure”requires skin graft

2x2 inch 4x10 inch 8x10 inch 5/pack

1. Used mainly in burns

Permanent Skin Substitute

The Role of Stem Cells

R. John Davenporteditor of Science’s SAGE KE

1 July 2005 Vol 309 SCIENCE www.sciencemag.orgPublished by AAAS Hematopoietic and stromal stem cell differentiation

Plasticity of adult stem cells

Br J Dermatol. 2005 Jul;153(1):29-36. Related Articles, Links

Human mesenchymal stem cells successfully improve skin-substitute wound healing.Nakagawa H, Akita S, Fukui M, Fujii T, Akino K.

Division of Plastic and Reconstructive Surgery, Department of Developmental and Reconstructive Medicine, Nagasaki University, Graduate School of Medical and Dental Sciences, 1-7-1 Sakamoto, Nagasaki 8528501, Japan.

About Stem Cells ResearchAbout Stem Cells Research

Severe BurnsSevere Burns

Severe burns are devastating injuries, Severe burns are devastating injuries, requiring long and painful recovery, and requiring long and painful recovery, and often resulting in significant scaring, often resulting in significant scaring, disfigurement and disability. Although disfigurement and disability. Although progress has been made with skin grafting progress has been made with skin grafting and artificial skin technologies, scientists and artificial skin technologies, scientists believe that stem cells could provide better believe that stem cells could provide better ways to regenerate functional skin ways to regenerate functional skin following burns.following burns.

For more information on burns, visit:• California Medical Association -- http://www.cmanet.org/• American Nurses Association of California -- http://www.anacalifornia.org/

• Human and Social Costs

According to the Sandia National Laboratories, there are over 100,000 burn victims annually in the U.S., receiving a total of almost one million in-hospital days spent in burn treatment. This represents approximately $2 billion in annual health care costs.

Potential for Stem Cell Therapies and Cures

Scientists have established that skin progenitor stem cells (called keratinocyte progenitors) in adult human skin have a significant capacity for growth and tissue-regeneration. It may also be possible to use embryonic stem cells to generate large numbers of healthy new epidermal or dermal skin cells.

Burn victims could also benefit from the stem cell therapy technique called somatic cell nuclear transfer, or SCNT. Using SCNT scientists can make large numbers of “patient specific” cells, meaning the cells’ DNA matches that of the original donor. Such cells could generate healthy new skin tissue grafts without the risk of the immune-rejection problems common to tissue and organ transplants.

Information obtained from the California Research and Cures Act.

About Stem Cells ResearchAbout Stem Cells Research

Potential for Stem Cell Therapies and CuresPotential for Stem Cell Therapies and Cures

Scientists have established that skin progenitor stem Scientists have established that skin progenitor stem cells (called keratinocyte progenitors) in adult human cells (called keratinocyte progenitors) in adult human skin have a significant capacity for growth and tissueskin have a significant capacity for growth and tissue--regeneration. It may also be possible to use embryonic regeneration. It may also be possible to use embryonic stem cells to generate large numbers of healthy new stem cells to generate large numbers of healthy new epidermal or dermal skin cells. epidermal or dermal skin cells.

Mesenchymal Bone Marrow Stem Cells More Effectively Stimulate

Regeneration of Deep Burn Wounds than Embryonic

Fibroblasts

V. I. Shumakov1, N. A. Onishchenko1, M. F. Rasulov1, M. E. Krasheninnikov1 and V. A. Zaidenov1

(1) Institute of Transplantology and Artificial Organs, Ministry ofHealth of Russian Federation, Moscow

Volume 136, Number 2 / August, 2003Available in website: http://www.springerlink.com/content/1wlqwjpqleqt/

Structure of regenerative epidermal-dermal equivalents based on EDC-collagen after one week (original magnification 200x, H&E staining). hMSCs promote stratification ( ) and proliferation of keratinocytes and result in a fully differentiated multilayered epidermis with organisation of rete ridge-like structures ( ).

Structure of regenerative epidermal-dermal equivalents based on EDC-collagen after one week (original magnification 200x, H&E staining). Keratinocytes seeded alone on the collagen matrix invaded intothe spongy structure ( ) and formed only a thin, irregular epidermal layer.

Conclusion

The approach to skin modelling reported here showed that non-skin-localized hMSC canpromote skin regeneration. The work suggests that direct intercellular contact is required for a skin-specific morphology. Co-cultures of hMSCs and keratinocytes may improve the performance of composite skin grafts in clinical applications

Stem cells of the skin epithelium Laura Alonso, and Elaine Fuchs *

Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10021

COLLOQUIUM PAPERS

Tissue stem cells form the cellular base for organ homeostasis and repair. Stem cellshave the unusual ability to renew themselves over the lifetime of the organ while producing daughter cells that differentiate into one or multiple lineages. Difficult to identify and characterize in any tissue, these cells are nonetheless hotly pursued because they hold the potential promise of therapeutic reprogramming to grow human tissue in vitro, for the treatment of human disease. The mammalian skin epitheliumexhibits remarkable turnover, punctuated by periods of even more rapid production after injury due to burn or wounding. The stem cells responsible for supplying this tissue with cellular substrate are not yet easily distinguishable from neighboring cells. However, in recent years a significant body of work has begun to characterize the skin epithelial stem cells, both in tissue culture and in mouse and human skin. Some epithelial cells cultured from skin exhibit prodigious proliferative potential; in fact, for >20 years now, cultured human skin has been used as a source of new skin to engraft onto damaged areas of burn patients, representing one of the first therapeutic uses of stem cells. Cell fate choices, including both self-renewal and differentiation, are crucial biological features of stem cells that are still poorly understood. Skin epithelial stemcells represent a ripe target for research into the fundamental mechanisms underlying these important processes.

Columnar organization of the epidermis

From Gambardella and Barrandon. Curr opin cell biol 200

Cornified layers

Suprabasal layersBasal layers

Dermis

Ex vivo expansion of adult autologous epidermal stem cells

1-5 cm2

1 m2

Fro Howard Green and colleagues(Rheinwald and Green, 1975. Gallico et al., N. Engl. J. Med, 1984)

Normal skin

Spontaneous healing

Epidermis generated from transplanted Stem cells

Regeneration of superficial dermis•Undulated dermo-epidermal junction• Presence of subepidermal vascular arcades•Presence of elastic fibers

•Observed in fetal wound healing•Never observed in post natal wound healing

Regeneration of epidermis•Normal keratinized epithelium•Presence of holoclones

Absence of epidermal appendages•Sweat glands, sebaceous glands, hair follicle

Why no epidermal appendages ?

1. Absence of multipotent epidermal stem cells

• No multipotent stem cells in adult skin• Multipotent stem cells do not survive in culture• Current culture conditions favor epidermal

differentiation

2. Absence of inductive signal(s)

The information remain scanty

• Difficulties to conduct experiment in human– Obvious ethical reasons– Regulatory rules (GMP)– Cost

• Difficulties regarding patient follow up • Poor communication between basic and medical

research laboratories• Difficulties to assay stemness• No control of stem cell engraftment• Necessity of a reliable and predictable animal model

Therapeutic use of skin stem cells Challenges