Gradients in lacunar morphology and cartilage mineralization reflect the mechanical function of the mouse femoral head epiphysis.

Mouse femurs are widely used to study bone development and disorders. The mammalian femoral head epiphysis, located between articular cartilage and a growth plate, critically maintains joint integrity during weight-bearing and supports femoral growth. Murine femoral head epiphyses are unusual in having no secondary ossification center (SOC). In this regard, a key question arises: How is the extracellular matrix (ECM) of the mouse femoral head epiphysis structured to balance the competing demands of mechanical stability and nutrient transport in the absence of a SOC? This study investigates the microstructure and ECM organization of normal young mouse femoral head epiphyses across multiple length scales and identifies distinct gradients in lacunar size, shape, mineral content, and collagen and mineral organization. Chondrocyte lacunae in deep epiphyseal zones are significantly larger, more spherical and interconnected, compared to the lacunae near the tidemark and growth plate. Enlarged lacunae and increased tissue porosity in the deep zones are associated with higher ECM mineralization, compensating for reduced stiffness from the porosity while maintaining compliance that may facilitate fluid flow and nutrient diffusion to enlarged cells. This study highlights an optimization strategy of murine proximal femoral epiphyses driven by mechanical and biological demands and it offers insights for designing engineered constructs. STATEMENT OF SIGNIFICANCE: The mouse femoral head epiphysis lacks a secondary ossification center (SOC) and is instead entirely comprised of calcified cartilage at a young age. Given that the SOC is thought to be essential for joint function in mammals, a key question arises: How does the young mouse femoral head epiphysis sustain chondrocyte viability while supporting mechanical function? Using multiscale 3D structural characterization, we identify unique gradients in chondrocyte lacunar morphology and extracellular matrix (ECM) organization. Our findings reveal a finely tuned balance between porosity-driven nutrient transport and mineralization-enhanced mechanical stability, offering novel insights into cartilage biology and functionality. These structural principles provide a foundation for biomimetic scaffold design in regenerative medicine, making this work highly relevant to the field of biomaterials and orthopedic tissue engineering.