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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:

Stop bleeding through clot formation. Release of inflammatory cytokines to regulate blood flow.

Recruitment of lymphocytes and macrophages.

Stimulate angiogenesis and collagen deposition (granulation tissue).

PDGF and TGF recruits actin-rich myofibroblasts to contract wound.

Keratinocyte stimulated

Granulation tissue resorbed leaving scar.

Categories of skin replacements:

An issue of cellular component




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:

1 X 1 cm skin sample from unburned site

Separation of dermis and epidermis

Culture on 3T3 feeder layer

Treat with dispase

Separate intact sheets

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:

Reduced donor sites in burn wounds.

Reduced wound contracture and scarring.

Quick closure (epithelialisation) of large acute excisional wounds.

Reduced treatment costs and hospital stay

Overcome problems associated with immunogenecity or the potential for BSE transfer.

Pharmaceutical applications such as human living skin equivalent in toxicity tests.

Current status in Thailand:

A survey on 140 hospitals in 69 provinces.

100% using autograft technique. A few using amnion (6%), allograft (5%) or commercial skin substitutes (5%).

88.1% aware of cultured skin graft.

92% willing to use the new technique.

Cost is the most concerned issue.

Cost of cultured skin sheets:

B 1400 for skin sheet patch of 58 cm2.

B 98000 for average patient with 22% total burn area. (B 720000-2600000 overseas)

B210000 to cover all medical costs. (B 160000 for conventional method)

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.