Graduation Date

Spring 5-7-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Molecular Genetics & Cell Biology

First Advisor

Mark A. Carlson

Abstract

Impaired tissue repair and regenerative ability in the aged is a major socio-economic and clinical problem. One of the likely factors associated with poor wound healing is the presence of lingering senescent cells in the wound sites. Failure to eliminate these cells has been associated with inadequate healing, excessive scarring, and abnormal skin stiffening. Thus, the objective of our study was to test interventional strategies to mitigate the negative effect of senescent cells. We hypothesized that reducing senescent cell burden and promoting non-senescent cell functionality will improve wound healing with age.

We developed a 3D in vitro senescent fibroblast populated collagen matrix (FPCM) to study the effect of chronic/persistent senescence on wound healing. A therapeutic approach with Nicotinamide (NAM) supplementation or a combination of a BCL-2 inhibitor (ABT-737) and FGF2 was used to analyze the effect on wound healing. We also tested the efficacy of our treatments in ex vivo human skin biopsy models obtained from healthy middle-aged donors. Additionally, we tested adipose-derived mesenchymal stem cells' effectiveness (AdMSC) in attenuating senescence and promoting healing in the in vitro and ex vivo skin biopsy models.

Data from the stress-induced senescent FPCM showed increased senescent cells with replicative, oxidative, or DNA damage-induced stress and a decline in wound healing efficacy with senescence induction. Treatment of the senescent FPCM with ABT-737 reduced the fraction of senescent cells from the matrices and accelerated wound healing rate. Combined ABT-737 and FGF2 treatment further improved the wound healing efficacy in the senescent models. Also, we observed a decline in NAD+ level with senescence, and supplementation with NAD+ precursor, NAM, increased the cellular NAD+ level and rescued the delayed healing response in the in vitro senescent model. In ex vivo explants, the percentages of wound closure were 85% with combined ABT-737/FGF2 treatment, 75% with NAM treatment, and 27% with media treatment alone. AdMSCs improved healing efficacy in vitro, as well as in the ex vivo skin biopsies by 75%. Taken together, we showed the reduction of these senescent cells with soluble factors improved the healing outcome in our in vitro and ex vivo models of healing.

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