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Reduction Of Senescent Cells Improves Healing In An In Vitro Model Of Aged Healing
Upasana Niyogi, Mark A. Carlson.
University of Nebraska Medical Center, Omaha, NE, USA.

BACKGROUND: One of the likely factors associated with poor healing in the aged is the increase of senescent cells in the wound. Since fibroblasts are one of the key players in wound healing, we utilized the fibroblast-populated collagen matrix (FPCM) with near-senescent human dermal fibroblasts in order to study the role of fibroblast senescence on wound healing. Senescent cells have been demonstrated to be resistant to apoptosis secondary to increased expression of BCL-2 proteins. So, we hypothesized that BCL-2 inhibition along with a mitogen stimulation would improve the wound healing endpoints in the senescent FPCM model.
METHODS: Senescence in the fibroblasts was induced either by replicative stress (RS) or oxidative stress (OS). Characterization of senescence was performed with fibroblast morphology assessment, senescence-associated beta-galactosidase activity (SA-▀Gal), proliferation assays, migration assays, and expression of senescence and oxidative stress markers. Wound healing ability of the young (passage < 10) vs. senescent fibroblasts was analyzed by monitoring the time required to heal scratch wounds made in the FPCM. The senescent FPCM was treated with a BCL-2 inhibitor (ABT-737) or a combination of ABT-737 and FGF-2 (fibroblast growth factor-2), followed by the scratch healing assay.
RESULTS: A delay in the wound healing associated with an increase in ▀-gal activity and expression of senescent cell markers was observed in the senescent FPCM compared to young the FPCM (p<0.0001). Treatment of the senescent FPCM with ABT-737 reduced the fraction of senescent cells and accelerated the scratch wound healing rate (p<0.005). Combined treatment with ABT-737 and FGF-2 further improved the healing rate in the senescent FPCM (p<0.003), reduced the population of alpha-SMA positive cells, and decreased collagen secretion, consistent with an anti-fibrotic effect.
CONCLUSIONS: Taken together, accumulation of senescent cells with age was associated with decreased healing efficacy in our aged in vitro model. Reduction of these cells with soluble factors improved healing outcomes in the model. We intend to test our therapeutic approach on wound healing in aged rodent models.


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