Pressure ulcers appear as localized chronic wounds in the middle of normal functioning skin. This study focuses on pressure ulcer fibroblasts cultured from the ulcer bed, ulcer margin, and normal, nonulcerated skin adjacent to the wound. From these areas we show that pressure ulcer fibroblasts become prematurely senescent. Verification of senescence was based on failure of cell populations to undergo a 0.5 population doubling after 1 week in culture, light microscopic appearance of late-passage senescent cells in culture, light microscopic verification of senescence in vivo and in vitro by anti-terminin staining, and ultrastructural identification of senescent fibroblasts by anti-terminin colloidal gold labeling. Although not all ulcer fibroblasts are senescent, there is a range of proliferative ability. The senescent phenotype of ulcer fibroblasts remains intact in vitro as these cells remain viable but are unable to complete DNA synthesis. Fibroblast senescence is not uniform in chronic wounds but varies within areas of the ulcer bed and from patient to patient. Although ulcer fibroblasts exhibit limited proliferative ability, fibroblasts from the ulcer margin and adjacent normal skin show a continued ability to divide. However, in all areas of the wound, the mean generation time of the fibroblast population is longer than commonly observed for cultured human diploid fibroblasts. At first passage, the mean generation time for fibroblasts from all patients is significantly different (p = .001, analysis of variance) among fibroblasts from the ulcer bed (4.2 2.2 days), ulcer margin (2.9 +/- 1.3 days), and adjacent normal skin (1.9 +/- 0.6 days). Analysis of the three cell groups by the post hoc Tukey test, using corrected p values shows that the difference between mean generation times among fibroblast populations from adjacent normal skin and ulcer bed are significantly different (p < 0.05), whereas the difference between fibroblasts from adjacent normal skin and ulcer margin and ulcer margin and ulcer bed are not significantly different (p > 0.05). These data may help to explain the poor response of certain pressure sores to aggressive medical treatment.

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p< 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.