The use of Stem cells in burn care
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  • 1. Introduction Burn • Tissue disruption due contact to source: thermal The use of Stem cells (burn/scald), chemical, electrical (lightning), radiation. in burn care • Problems encountered: – Acute phase • Deterioration of airway, breathing and circulation • Systemic Inflammatory Response Syndrome Yefta Moenadjat (SIRS) & Multi-system Organ Dysfunction Syndrome (MODS) – Late phase • Wound healing Introduction Superficial burn Burn • Epidermal layer disruption • Intact dermal • Tissue disruption: wound classification • Painful • Spontaneous healing 5-7 Type of wound days 1 Superficial burn 1o • Treatment: 2 Partial thickness burn 2o • Moisturizer cream • Common analgesic • superficial partial 2o superf thickness • deep partial thickness 2o deep (full thickness) 3 Deep burn 3o Partial thickness Partial thickness burn burn 1. Superficial 2. Deep (full thickness) • Epidermal layer disruption • Epidermal layer disruption • 1/3 superficial dermal layer • 2/3 superficial dermal layer • Blister formation • Blister formation (+/─) • Painful • Thin eschar • Intact dermis > • Painful Spontaneous healing 10-14 • Intact skin appendices days Spontaneous healing up to 21 • Treatment: days • Moisturizer cream • Treatment: • Blister management • Moist dressing • Common analgesic • Blister management • Common analgesic
  • 2. Débridement: Deep burn Surgical Non surgical • Epidermal layer, dermal layer Concept changes: and deeper layer (subcutaneous • Total excision Second place & adipose tissue, muscles, • Tangential excision bones) • Total excision • Eschar Rapid Slow • No sensation Complication: No bleeding • Intact dermis & skin appendices Surgical bleeding complication (─) Method: Spontaneous healing impossible • Conventional (Humby Method: • Treatment: knife) • Autolytic • Debridement (escharectomy) • Electric dermatome • Enzymatic • Skin grafting • Hydropressure Debris removal referred to source control The wound closure Wound healing: Review Phases of Wound healing • Problems encountered in burn wound closure Homeostasis & inflammation Maturation • Degree of severity Fibroplasia – Damaged tissue → deteriorated circulation→ non vital tissue → inflammatory response ▲ – Burn exhaustion [metabolic changes, inflammation] – Prolonged phases of wound healing • Impaired – non healing wound 0 2 4 6 8 365 Days after injury Injury Phase of proliferative [fibroplasias, fibro-proliferative] Phase of Fibroplasias • Template formation – Proliferation of fibrin cells [collagen matrix ] replaces the clot • Angiogenesis – Proliferation of endothelial cells [new vessels formation] ►Granulation tissue • Epithelialization – Proliferation of epithelial cells from wound edges and 1. Fibrin proliferation skin appendices early
  • 3. Phase of Fibroplasias 1. Fibrin proliferation early Phase of Fibroplasias Phase of Fibroplasias 1. Fibrin proliferation 1. Fibrin proliferation early late Phase of Fibroplasias Phase of Fibroplasias 1. Fibrin proliferation 2. Angiogenesis late early
  • 4. Phase of Fibroplasias Phase of Fibroplasias 2. Angiogenesis 2. Angiogenesis early late Phase of Fibroplasias Phase of Fibroplasias Granulation: Fibrin proliferation + Angiogenesis Granulation: Fibrin proliferation + Angiogenesis Phase of Fibroplasias Phase of Fibroplasias Wound: contraction 3. Epithelialization early
  • 5. Epithelialization: Phase of Fibroplasias Epithelization is start from the wound edges Basal membrane 3. Epithelialization complete Epithelialization: Epithelization is not always start from the wound edges Sweat gland Sebaseous gland Hair follicle Phase of remodeling [phase of maturation] Conditions of fibroplasias • Collagen deposition [early, 2 mo] – Indurative tissue • Healthy granulation • Collagen resorption [late, up to 8-12 mo] tissues – Soften tissue – Adequate collagen • Regression of vessels matrix – Tissue becomes pale – Angiogenesis ►Firm and pale tissue ►Scar tissue Granulation tissue
  • 6. Conditions of fibroplasias • Healthy granulation tissues – Adequate collagen matrix – Angiogenesis ►excessive granulation tissue Granulation tissue Influencing factors • Structural component or scaffolding • Biologically active component stimulating all phases of healing • Collagen (protein) – Scaffold for cell migration and matrix deposition – Cell guidance • Elastin (protein) – Tissue elasticity • Fibronectin (protein) – Cell to cell adherence – Contact orientation for cells – Increases epithelial cell division, migration – Chemo attractant for fibroblasts, macrophages Influencing factors • Growth Factors (proteins) • Stimulate all phases of wound healing • Glycosaminoglycan (glycosylated protein) – Cell adherence properties – Conduit for healing factors – Deactivator of proteases – Scaffold or foundation for dermal elements • Hyaluronic Acid (complex carbohydrate) – Maintaining matrix hydrated – Decreases inflammation – Stimulates healing – Proper cell alignment
  • 7. Temporary Skin Substitute • Injury SIRS and MODS Product Company Tissue of Origin Layers Category Uses How supplied • Inflammation • Inadequate blood flow Temporary Frozen in Split coverage of • Ischemia-reperfusion injury Ischemia- Human Human Epidermis and rolls of Skin bank thickness large allograft cadaver dermis varying • Infection skin excised size burns Temporary coverage Brennan Autolysis Pig dermis Dermis Dermis of partial Frozen or Toxins Infection Healing Pig skin Medical St. thickness refrigerated Inflammation Xenograft Louis, Mo and in rolls excised burns Epidermis ↑ demand for ↑ demand for ↑ demand for Human On site Placenta Amniotic Dermis Same as Refrigerato amnion procurement membrane above r Inflammatory cells Immune modulation Mesenchymal stem cells Room T° Extracellular Superficial Healthpoint Bioactive storage Oasis® Xenograft wound matrix burns Skin ↑ demand on LTD San from small Dermal like graft donor Multiple Marrow suppression Antonio, Tx Matrix sizes Bone Marrow support intestine sites 3x3.5cm submucosa Chronic 7x20cm wounds Marrow Exhaustion Temporary Skin Substitute Permanent Skin Substitute Tissue of How AVAILABLE PERMANENT SKIN SUBSTITUTES Product Company Layers Category Uses Origin supplied Tissue of How Product Company Layers Category Uses Origin supplied Superficial Synthetic Room T° Chronic Dow Bilayer Synthetic partial Biobrane® with added storage Composite wounds, Hickam/Bertek product outer epidermis thickness Collagen matrix 7.5cm denatured 15x20inch Organogenesis : often used Pharmaceutica silicone Inner and dermis burns,Temp seeded with diameter bovine 10x15cm Inc and Novartis Allogenic Epidermis with thin ls nylon mesh orary cover Apligraf human neonatal disc collagen 5x15inch Pharmaceuticals Composite and STSG with added of excised keratinocytes 1/pack 5x5 inch Corp Dermis Excised collagen burns and fibroblasts deep burn Superficial Bioactive to mid- Composite Bilayer Collagen sponge Skin graft Smith & Dermal Partial Ortec : 6x6cm Transcyte® Allogenic product Outer Frozen in OrCel Allogenic seeded with donor site, Nephew Matrix thickness International Epidermis sheets Dermis silicone Inner 5x7.5 inch Composite human neonatal chronic Wound Components burns Inc. and nylon seeded sheets keratinocytes wounds Management on Synthetic Temporary Dermis with neonatal and fibroblasts Largo, FL dermis and coverage of fibroblasts Deep epidermis excised burns partial and 50cm2 Cultured full Genzyme Tissue Autogenous Epidermis sheets in Epicel* autologous thickness Repair Corp keratinocytes Only culture keratinocytes burns medium >30% TBSA 1. Used mainly in burns Permanent Skin Substitute AVAILABLE PERMANENT SKIN SUBSTITUTES Tissue of How Product Company Layers Category Uses Origin supplied Deep partial and full A cellular thickness Allogenic Dermis Dermis 1x2cm to Alloderm Life Cell burns, Soft dermis (processed only 4x12cm tissue allograft) replacemen t, Tissue patches Full The Role of Stem Cells thickness 2x2 inch Silicone outer soft tissue 4x10 Integra Life layer on Biosyntheti defects inch Integra* Synthetic Science Corp collagen GAG c Dermis definitive 8x10 dermal matrix “closure” inch requires 5/pack skin graft 1. Used mainly in burns
  • 8. R. John Davenport editor of Science’s SAGE KE 1 July 2005 Vol 309 SCIENCE Published by AAAS Hematopoietic and stromal stem cell differentiation 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. Plasticity of adult stem cells About Stem Cells Research About Stem Cells Research • Human and Social Costs Severe Burns 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 Severe burns are devastating injuries, burn treatment. This represents approximately $2 billion in annual health care costs. Potential for Stem Cell Therapies and Cures requiring long and painful recovery, and Potential for Stem Cell Therapies and Cures often resulting in significant scaring, Scientists have established that skin progenitor stem cellsprogenitor stem Scientists have established that skin (called keratinocyte disfigurement and disability. Although cellsprogenitors) inItadult human skin haveto use embryonic stemfor growth and tissue- (called keratinocyte progenitors) in adult human regeneration. may also be possible a significant capacity cells to generate large progress has been made with skin grafting skin numbers of healthy new epidermal or dermal skin cells. have a significant capacity for growth and tissue- tissue- and artificial skin technologies, scientists regeneration. Italso benefit from the stem cell therapy technique called somatic Burn victims could may also be possible to use embryonic believe that stem cells could provide better stem cells to generateUsing SCNT scientists can makethe original donor. cell nuclear transfer, or SCNT. large numbers of healthy new “patient specific” cells, meaning the cells’ DNA matches that of large numbers of ways to regenerate functional skin epidermal coulddermalcommon cells. and grafts without the risk of the Such cells or generate healthy new skin tissue immune-rejection problems skin to tissue organ transplants. following burns. Information obtained from the California Research and Cures Act. For more information on burns, visit: • California Medical Association -- • American Nurses Association of California --
  • 9. 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, Structure of regenerative M. E. Krasheninnikov1 and V. A. Zaidenov1 epidermal-dermal equivalents based on EDC-collagen after (1) Institute of Transplantology and Artificial Organs, Ministry of one week (original Health of Russian Federation, Moscow magnification 200x, H&E Volume 136, Number 2 / August, 2003 staining). hMSCs promote Available in website: stratification ( ) and proliferation of keratinocytes and result in a fully differentiated multilayered epidermis with organisation of rete ridge-like structures ( ). Conclusion Structure of regenerative epidermal-dermal equivalents The approach to skin modelling reported here based on EDC-collagen after one week (original showed that non-skin-localized hMSC can magnification 200x, H&E promote skin regeneration. The work suggests staining). Keratinocytes seeded alone on the collagen that direct intercellular contact is required for a matrix invaded into skin-specific morphology. Co-cultures of the spongy structure ( ) and formed only a thin, irregular hMSCs and keratinocytes may improve the epidermal layer. performance of composite skin grafts in clinical applications COLLOQUIUM PAPERS 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 Tissue stem cells form the cellular base for organ homeostasis and repair. Stem cells have 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 epithelium exhibits 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 stem cells represent a ripe target for research into the fundamental mechanisms underlying these important processes.
  • 10. Columnar organization of the epidermis Cornified layers Suprabasal layers Basal layers Dermis From Gambardella and Barrandon. Curr opin cell biol 200 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
  • 11. Regeneration of epidermis •Normal keratinized epithelium •Presence of holoclones 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 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
  • 12. Therapeutic use of skin stem cells Challenges