LECTURER
Lecture Topic: In vitro Culture of Human Skin Graft Sheets for Wound Therapy
Objectives: To set up an in vitro human keratinocyte culture system
To develop and make available lab grown skin grafts for domestic burn patients.
Structure of epithelium:
The epidermis is comprised primarily of keratinocytes which form a stratified squamous epithelium.
Normal wound healing process:
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Stop bleeding through clot formation. Release of inflammatory cytokines to regulate blood flow. |
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Recruitment of lymphocytes and macrophages. |
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Stimulate angiogenesis and collagen deposition (granulation tissue). |
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PDGF and TGF recruits actin-rich myofibroblasts to contract wound. |
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Keratinocyte stimulated |
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Granulation tissue resorbed leaving scar. |
Categories of skin replacements:
An issue of cellular component
Autologous
Allogenic
Xenogenic
Autografting techniques:
Classical skin graft:
Removal of patient ‘s own skin from donor sites. Accompanies morbidity, pain, scarring and sometimes insufficient.
Cultured keratinocyte sheet:
Large number of sheets can be grown from a small piece of patient’s normal skin.
Culture of human keratinocyte: experimental protocol:
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1 X 1 cm skin sample from unburned site |
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Separation of dermis and epidermis |
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Culture on 3T3 feeder layer |
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Treat with dispase |
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Separate intact sheets |
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Apply to wound |
Acquisition of initial skin biopsy sample:
Sample sources:
Fore skin: Children Hospital and Rama Hospital
Adult skin: Siriraj, Chula, Nopparat and others
Significant therapeutic benefits:
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Reduced donor sites in burn wounds. |
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Reduced wound contracture and scarring. |
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Quick closure (epithelialisation) of large acute excisional wounds. |
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Reduced treatment costs and hospital stay |
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Overcome problems associated with immunogenecity or the potential for BSE transfer. |
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Pharmaceutical applications such as human living skin equivalent in toxicity tests. |
Current status in Thailand:
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A survey on 140 hospitals in 69 provinces. |
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100% using autograft technique. A few using amnion (6%), allograft (5%) or commercial skin substitutes (5%). |
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88.1% aware of cultured skin graft. |
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92% willing to use the new technique. |
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Cost is the most concerned issue. |
Cost of cultured skin sheets:
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B 1400 for skin sheet patch of 58 cm2. |
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B 98000 for average patient with 22% total burn area. (B 720000-2600000 overseas) |
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B210000 to cover all medical costs. (B 160000 for conventional method) |
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Length of stay 30-35% shorter |
Tissue engineering of the skin:
Providing or mimicking structural and/or biological characteristics of the dermal or the epidermal component.
Most are combinations of appropriate scaffolds (templates) and living cells.
Skin substitute approach:
Cellular Component(Organotypic culture):
May consists of type I collagen gel seeded with allogenic fibroblasts (from foreskin biopsies) covered with a confluent surface layer of allogenic keratinocytes. This approach has some benefit as viable allogenic cells may deliver biological mediators (growth factors such as TGF-beta and IL-1) capable of accelerating repair process. Needs organotypic culture technique.
Supporting matrix component (seeded device):
Polyglycolic acid, polyglactin-910, chitosan, glycosaminoglycans (chondroitin-6-sulphate, hayaluronic acid etc), silk protein fibroin. These materials have wide surgical usage ( and acceptance) as bioresorbable sutures and have been variously applied in clinical usages. Anti-bacterial property is favourable.