REGENERATION PATTERN OF BLOOD-VESSELS AND NERVES IN CULTURED KERATINOCYTE GRAFTS ASSESSED BY CONFOCAL LASER-SCANNING MICROSCOPY

The aim of this study was to investigate the pattern of both neovascul arization and reinnervation, and the relationship between the two proc esses, in keratodermal grafts, using confocal laser scanning microscop y, at different time points during the healing process. Keratodermal g rafts were prepared in pigs by combining autologous dermis with cultur ed autologous keratinocytes. Immunohistochemistry was carried out on t hick cryostat sections (100-150 mu m), using antisera to the endotheli al marker von Willebrand factor (vWf) and the pan-neuronal marker prot ein gene product 9 . 5 (PGP9 . 5). The results suggest that the neovas cularization and reinnervation in the cultured keratodermal graft is a lmost complete at 6 weeks. Neovascularization precedes innervation, re aching the surface covered by the keratinocytes at 2 weeks, initially with a linear vascular pattern. From 3 weeks, there is a gradual arbor ization of the vessels to form a typical vascular plexus. The process of reinnervation is similar in pattern to that of neovascularization, although slower in developing a full network of fibres. In conclusion, the use of confocal microscopy allows the precise definition of compl ex patterns of neovascularization and nerve growth, which are not full y apparent when using conventional microscopy. Because angiogenesis oc curs first, it probably plays a leading role in the survival of kerato dermal grafts during wound healing. Indeed, new blood vessels form a p athway for the subsequent innervation process, and quickly reach the e pidermal layer which, in turn, may play a key role in the tropism of b oth blood vessels and nerves.