Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury.
The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.
Wright
K A
KA
Genzyme Tissue Repair, Cell Biology Group, Framingham, MA 01710-9322, USA.
Nadire
K B
KB
Busto
P
P
Tubo
R
R
McPherson
J M
JM
Wentworth
B M
BM
D016475:3T3 Cells / Q000166:cytology; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D001458:Bandages; D002056:Burns / Q000601:surgery*; D002118:Calcium / Q000008:administration & dosage; D002448:Cell Adhesion; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D046508:Culture Techniques; D004848:Epithelium / Q000502:physiology; D000939:Epitopes / Q000032:analysis; D005240:Feasibility Studies; D006801:Humans; D039161:Integrin alpha6beta4; D016023:Integrins / Q000032:analysis; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D007797:Laminin / Q000032:analysis; D008567:Membranes, Artificial; D051379:Mice; D008819:Mice, Nude; D014677:Pharmaceutical Vehicles; D011140:Polyurethanes; D011498:Protein Precursors / Q000032:analysis; D012867:Skin / Q000166:cytology / Q000293:injuries / Q000473:pathology; D013045:Species Specificity; D014182:Transplantation, Autologous
D000954:Antigens, Surface; D003470:Culture Media; D000939:Epitopes; D039161:Integrin alpha6beta4; D016023:Integrins; D007797:Laminin; D008567:Membranes, Artificial; D014677:Pharmaceutical Vehicles; D011140:Polyurethanes; D011498:Protein Precursors; C032253:involucrin; D002118:Calcium