Fiber density–driven mechanotransduction reconstructs collagen heterogeneity in the annulus fibrosus

Existing annulus fibrosus (AF) repair strategies largely restore structural continuity without reconstructing native collagen heterogeneity, due to limited control over annulus fibrosus cell (AFCs) phenotypes. Here, we hypothesize that fiber density-mediated mechanical cues can directionally regulate AFCs collagen specification. Three‑dimensional electrospun poly(lactic acid)/gelatin fibrous scaffolds with identical composition but distinct fiber densities were fabricated to generate low‑density (LDS) and high‑density (HDS) scaffolds. Fiber density exerted a decisive influence on AFCs phenotype through differential mechanotransduction. LDS was associated with increased collagen type I and α-smooth muscle actin expression, together with elevated RhoA-ROCK-related gene expression and enhanced ERK/AKT phosphorylation, consistent with a fibroblastic phenotype. In contrast, HDS favored collagen type II (COL-II) and aggrecan expression and was associated with increased Piezo1 expression; pharmacological inhibition of Piezo1-related mechanosensing attenuated HDS-associated COL-II expression, supporting its partial contribution to the cartilaginous-like phenotype. Subcutaneous implantation in rats further demonstrated fiber density-dependent differences in host cell infiltration, collagen deposition, and tissue integration in an ectopic environment. Collectively, this study identifies fiber density as an important mechanobiological design parameter for regulating AFCs phenotype and matrix remodeling, providing a structural basis for future AF repair strategies. STATEMENT OF SIGNIFICANCE: Recreating the region-specific collagen heterogeneity of the annulus fibrosus (AF) is a fundamental yet unmet challenge in intervertebral disc regeneration, as this structural hierarchy is essential for anisotropic mechanical function. Current scaffold-based strategies largely fail to provide spatially tunable mechanical cues to direct cell phenotype. This study introduces a biomaterial strategy using three-dimensional scaffolds with programmed low- or high-density fiber microarchitectures. The results showed that low-density scaffolds were associated with a COL-I-enriched phenotype and increased RhoA-ROCK- and MAPK/ERK-related signaling, whereas high-density scaffolds were associated with a COL-II-enriched phenotype and enhanced mechanosensitive Ca²⁺-related signaling. These findings suggest that scaffold fiber density may serve as a useful microarchitectural design parameter for modulating AF cell matrix phenotypes. This study provides a reference for the future design of spatially organized scaffolds aimed at supporting heterogeneous collagen matrix reconstruction in AF repair.