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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