Transient Senescence Drives Regenerative Niche Formation: Premature Senolytic Clearance Disrupts Fibroblast‑Derived Morphogenetic Signaling in Acute Wound Healing

Hypothesis

Transiently generated senescent fibroblasts in acute wounds create a regenerative niche by secreting morphogenetic factors that guide neighboring stem cells; removing these cells with senolytics during the early repair phase impairs this signaling and delays tissue restoration.

Mechanistic Rationale

Young wound fibroblasts that become p16+/p21+/Mki67− senescent activate developmental gene programs enriched for morphogenetic pathways [1]. These cells secrete a distinct SASP fraction rich in fibroblast growth factors, Wnt modulators, and matrix remodeling enzymes that collectively establish a permissive microenvironment for epithelial and stem‑cell proliferation. In contrast, chronic senescent cells produce a proinflammatory SASP that drives fibrosis. The temporal switch from a regenerative to a deleterious SASP is governed by intracellular NAD+ levels and chromatin accessibility at enhancers of SASP genes; acute injury transiently lowers NAD+, favoring a benign SASP, while persistent oxidative stress maintains high NAD+ and locks in a harmful profile.

Testable Predictions

1. Senolytic treatment administered within 24 h post‑injury will reduce the proportion of p16+/p21+/Mki67− fibroblasts in the wound granulation tissue by >50 % compared with vehicle.
2. This reduction will correlate with a measurable decrease in wound‑area closure rate at day 7 (∼30 % slower) and diminished expression of morphogenetic markers (e.g., Fgf7, Wnt5a) in the surrounding tissue.
3. Exogenous addition of the putative regenerative SASP fraction (isolated from young senescent fibroblast conditioned medium) will rescue the healing deficit caused by early senolytic clearance.
4. Delaying senolytic administration until day 4 post‑injury (after the transient senescence peak) will not impair closure speed and will still reduce later‑stage proinflammatory SASP markers.

Experimental Design

• Use full‑thickness dorsal excisional wounds in 8‑week‑old C57BL/6 mice (n = 10 per group).
• Groups: (a) vehicle control, (b) senolytic (dasatinib + quercetin) given intraperitoneally at 0 h, (c) senolytic given at 48 h, (d) vehicle + recombinant regenerative SASP protein cocktail.
• Harvest wounds at 0 h, 24 h, 72 h, and 7 d for flow cytometry (p16, p21, Ki67), immunofluorescence (α‑SMA, collagen I), and qPCR for SASP components (Il6, Cxcl1, Fgf7, Wnt5a).
• Monitor wound area daily via planimetry; calculate closure rate.
• Perform RNA‑seq on sorted fibroblasts to verify enrichment of developmental gene sets in early senescent cells and its loss after early senolytic treatment.

Potential Outcomes and Falsifiability

If early senolytic clearance significantly slows wound closure and reduces morphogenetic SASP factors, and if adding back the regenerative SASP rescues the phenotype, the hypothesis is supported. Conversely, if early senolytic administration does not affect closure kinetics or morphogenetic gene expression, or if the regenerative SASP fraction fails to ameliorate any defect, the hypothesis is falsified. This design directly tests the premise that transient senescent cells are necessary chaperones during acute regeneration, challenging the assumption that senolytic clearance is universally beneficial.