IF allogeneic bone marrow-derived mesenchymal stromal cells (MSCs; 1×10^6 cells per lobe, delivered via 29–30 gauge needle at a 30–45° angle under direct visualization following partial upper median sternotomy) preconditioned for 72 hours on decellularized aged (18-month-old C57BL/6 donor) thymic extracellular matrix scaffolds — prepared via three freeze-thaw cycles (−80°C/37°C) followed by sequential 1% Triton X-100 / 0.1% SDS detergent treatment and extensive PBS wash — are administered by intrathymic injection into 18–22 month-old C57BL/6 mice (both sexes, n=15 per group),

THEN at week 12 post-injection, the frequency of CD44^low CD62L^high cells among peripheral blood CD4^+ and CD8^+ T cells will reach ≥40% of 8-week-old reference controls (versus <20% in PBS vehicle controls), the splenic TCRβ CDR3 Shannon entropy index will increase by ≥0.3 units versus vehicle, and thymic cellularity at sacrifice will show a statistically significant increase in total thymocyte number versus aged vehicle — and critically, aged-ECM-preconditioned MSCs will outperform both PBS vehicle and naive (unconditioned) MSCs by ≥15 percentage points in naive T cell frequency recovery, with this superiority explained by the hypothesis that aged thymic ECM acts as a damage-specific molecular "blueprint" that calibrates the MSC secretome toward simultaneous anti-fibrotic and thymopoietic repair programming, an effect not recapitulated by naive MSCs or — as proposed in the novel extension — by MSCs conditioned on young thymic ECM,

BECAUSE the following step-by-step causal chain connects the intervention to the outcome:

1. Aged thymic ECM encodes a damage-specific biochemical landscape distinct from young ECM. Advanced thymic involution in 18-month C57BL/6 mice is characterized by progressive fibrotic remodeling, adipocyte infiltration, and an altered matricellular protein composition — including shifted collagen IV/laminin ratios and TGF-β1 sequestration within the matrix — representing accumulated structural damage that cannot be reversed by systemic interventions alone. The decellularized aged thymic ECM therefore presents a molecular damage signature that is physically absent from both young thymic ECM and from plastic culture substrates. (Aged ECM damage signatures and collagen remodeling in thymic involution are described in the synthesized evidence — see ECM Scaffold Preparation section of the Evidence Set; Crapo et al., Biomaterials, 2011, referenced in Evidence Set for general decellularization benchmarks.)

2. Seventy-two-hour preconditioning on aged thymic ECM instructs MSCs through mechanosensing and matrikine signaling to adopt a repair-specific secretory phenotype. MSCs cultured on decellularized ECM scaffolds undergo substrate-driven transcriptional reprogramming via integrin-FAK-YAP/TAZ mechanotransduction pathways. On aged thymic ECM specifically, the stiffer, more fibrotic substrate — compared to young ECM — is predicted [SPECULATIVE]...

SENS category: GlycoSENS