Hypothesis

Transient senescent fibroblasts act as chaperones that secrete a specific SASP signature (CCL2, IGF‑1, and matrix‑remodeling enzymes) essential for the proliferative phase of wound healing, whereas chronic senescent fibroblasts switch to a TGF‑β1‑dominant SASP that drives fibrosis. A digital twin integrating longitudinal serum biomarkers (IL‑6/TGF‑β ratio, circulating p16^INK4a^+ and p21^+ extracellular vesicles) and individual injury dynamics can predict the temporal switch from reparative to pathogenic senescence, enabling marker‑selective senolytic clearance (p21^low/p16^high^) only after the chaperone phase has ended.

Mechanistic Basis

Recent work shows that acute senescent cells in wounds secrete PDGF‑AA, IL‑6, and CCN1/CCN2 to recruit immune cells and promote tissue remodeling [1]. We propose that, in addition to these factors, a transient senescent fibroblast subset releases CCL2‑rich exosomes that polarize macrophages toward an M2‑like phenotype, which then secretes IGF‑1 to stimulate keratinocyte and fibroblast proliferation. As senescence persists, epigenetic remodeling shifts the SASP toward TGF‑β1, SMAD2/3 activation, and increased LOX‑mediated collagen cross‑linking, culminating in a stiff, fibrotic matrix [3]. The digital twin would detect the rising serum TGF‑β1/IL‑6 ratio and an increase in p16^INK4a^+ extracellular vesicles as a signature of the pathogenic switch.

Testable Predictions

1. In murine full‑thickness skin wounds, early senolytic treatment (day 1‑post‑injury) will reduce wound tensile strength and delay re‑epithelialization compared with vehicle, despite lowering p16^INK4a^+ cell numbers.
2. Delayed senolytic administration guided by a digital twin algorithm (initiated when the serum TGF‑β1/IL‑6 ratio exceeds a threshold of 2.5 and p16^INK4a^+ EVs rise >30 % above baseline) will preserve early CCL2^+ senescence, enhance macrophage M2 polarization, and result in faster wound closure and stronger scar tissue than either untreated or early‑cleared groups.
3. Genetic ablation of CCL2 in senescent fibroblasts will phenocopy the deleterious effects of early senolysis, confirming the chaperone role of this SASP component.

Experimental Design

• Use aged (20‑month) C57BL/6 mice with dorsal excisional wounds.
• Administer navitoclax (a Bcl‑2 family senolytic) or vehicle at three time points: (i) day 1, (ii) day 4 (predicted by twin), (iii) day 7 (late chronic phase).
• Collect blood daily for IL‑6, TGF‑β, and EV‑associated p16^INK4a^/p21^ quantification.
• Harvest wounds at days 4, 7, and 14 for histology (α‑SMA, collagen I), immunofluorescence for CCL2^+ senescent fibroblasts, flow cytometry for macrophage phenotypes, and biomechanical testing.
• Construct a digital twin for each animal using the biomarker trajectories to predict the optimal clearance window.

Potential Outcomes and Falsifiability

If early senolysis impairs healing and twin‑guided delayed senolysis improves it, the hypothesis is supported. If early senolysis shows no detriment or twin‑guided timing confers no advantage over fixed‑delay senolysis, the hypothesis is falsified, indicating that senescent cells do not provide a necessary chaperone signal or that the biomarker signature does not accurately reflect the functional switch.