During the reepithelialization of cutaneous wounds, the migratory epidermis transits over a provisional matrix of fibronectin and fibrin in the absence of laminin and type IV collagen as well as ultrastructurally identifiable basement membrane. Since significant quantities of fibronectin occur at these sites of reepithelialization, we surmised that fibronectin is a suitable substrate for keratinocyte adherence and therefore undertook the in vitro investigations reported here. Purified human plasma fibronectin precoated on bacteriologic microtiter wells was demonstrated to mediate human keratinocyte adherence when concentrations greater than 10 micrograms/ml fibronectin were used. Maximal keratinocyte adherence was obtained in wells precoated with 100 micrograms/ml fibronectin and when cells were incubated with substrate for 60 min or longer at 37 degrees C. Both primary and second-passaged human keratinocytes adhered as well or better to fibronectin than to types I and III collagen, laminin, or type IV collagen under both high- and low-Ca++ culture conditions. However maximal adherence to all substrates occurred when second-passaged keratinocytes were assayed in low-Ca++ medium. Under these latter culture conditions, keratinocyte phenotype resembles the phenotype of cells in the migrating epidermis. To determine specificity of these adherence reactions, antifibronectin antibodies were shown to block keratinocyte adherence to fibronectin but not to laminin substrates. Conversely, antilaminin antibodies blocked adherence to laminin but not fibronectin substrates. Thus, human keratinocytes demonstrate specific adherence to fibronectin in a time- and dose-dependent fashion and this adherence relies on de novo protein synthesis. We take these observations to support our hypothesis that the provisional fibronectin matrix observed beneath the migrating epithelium during tissue repair plays a functional role in the reepithelialization process.

Myotonic dystrophy (MyD) has been suggested to be a segmental progeroid syndrome in man, as this syndrome has some clinical manifestations of premature aging. Fibroblasts from patients with other progeroid syndromes have been shown to have diminished in vitro lifespans or growth characteristics; therefore, it was of interest to study cellular senescence in fibroblasts from patients with MyD. Fibroblast cultures from patients with Duchenne muscular dystrophy (DMD) were used as additional controls, as premature aging is not associated with this genetic disorder. Primary skin fibroblast cultures obtained from patients with MyD or DMD and from age-sex matched controls were grown in DMEM plus 10% FBS. The in vitro lifespan was determined by either a 1:4 split ratio or with a constant initial inoculum of 1 times 10(4) cells/cm2, followed by determination of the final density at weekly intervals. Our results demonstrate that there is no difference in the limits of the in vitro lifespan for either the MyD or DMD fibroblast strains compared to the controls. Likewise, no difference could be detected in the growth characteristics of these cells. The only observable difference was that the pooled age-matched controls and MyD cultures had a shorter in vitro lifespan than the DMD group and their pooled controls, a finding expected because of the age of the patients in each group. Unlike the other progeroid syndromes, MyD fibroblasts have normal limits for in vitro lifespan. MyD is probably not closely related to the other premature aging syndromes, although there is an increasing phenotypic expression as a function of age.

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Left hind limbs, including the pelvis, of adult newts (Notophthalmus viridescens) were locally irradiated with a dose of x-rays that inhibited regeneration (2,000 R). This x-ray dose and other doses (700-2,000 R) capable of inhibiting limb regeneration also cause limb regression prior to amputation. Before limb regression occurred, there was a latent period of 3 to 6 weeks. Limb regression was characterized by necrotic wasting and resorption of distal elements. The degree of loss was variable and dependent upon dosage. After this further degenerative changes were not noted. Proliferation of epidermal cells was examined 4 days after irradiation prior to limb regression or after x-ray-induced degeneration of the limbs had ended. Proliferative activity in x-rayed limbs was also compared at various stages of contralateral control limb regeneration. Limbs examined after x-ray-induced limb regression had ended showed levels of [3H]-thymidine incorporation into DNA comparable to normal epidermis. In contrast, limbs examined 4 days after irradiation had lower levels of DNA synthesis (P much less than 0.01). Amputation of limbs in both groups caused an increase in DNA synthesis (P much less than 0.01). Histological examination showed that cellular proliferation was associated primarily with the epidermis. These results indicate that epidermal cell proliferation was not resistant to x-rays. However, levels of normal cell division were observed after amputation of after cessation of x-ray-induced limb regression.