Effect of keratinocyte seeding of collagen-glycosaminoglycan membranes on the regeneration of skin in a porcine model

A collagen-glycosaminoglycan matrix, impregnated with autologous keratinocytes, was applied as island grafts onto full-thickness porcine wounds to determine whether complete epidermal coverage could be achieved in a single grafting procedure. Twenty-four grafts with seeding densities ranging from 0 to 3,000,000 cells/cm² were used to determine the kinetics of epidermal coverage. The time sequence of epidermal formation was then studied between days 14 and 28 using four additional grafts, each seeded with a density of 500,000 cells/cm². Autologous keratinocytes proliferated as the collagen-glycosaminoglycan matrix was vascularized to form a confluent epidermis by 2 weeks in matrices seeded with at least 100,000 cells/cm². The epidermal thickness and the number of keratinocyte cysts observed in the neodermis at 2 weeks increased linearly with the logarithm of the seeding density. Sequential analysis of neoepidermis showed the nascent epidermis to be hyperplastic, parakeratotic, and focally lacking in granular layer differentiation at 2 weeks. After 2 weeks, it underwent normal maturation and differentiation. Irrespective of seeding density at 2 weeks the collagen- glycosaminoglycan matrix was well vascularized, contained a dense cellular infiltrate, and was almost completely degraded. These studies demonstrate that seeded keratinocytes proliferate and differentiate to form a confluent epidermis by 2 weeks in matrices seeded with at least 100,000 cells/cm².