Bone tissue regeneration in critical sized defects presents a currently intractable challenge in orthopaedics. New approaches seek to harness or replicate the regenerative capacity of the periosteum, the tissue membrane bounding bone that provides a niche for mechanosensitive osteochondral progenitor cells. Yet periosteum's unique properties are not well understood across spatiotemporal scales or from a structure-function perspective. This thesis presents multiple length- (molecular, cellular, tissue) and time-scale (growth, aging, lifetime) approaches to elucidate periosteum’s structure-function relationships, while addressing key considerations for optimizing a clinically translatable periosteum substitute. Beginning at a tissue scale, a biomechanics model of a virtual human femur simulates the emergence of the linea aspera, a characteristic, anatomic ridge along the posterior aspect of the femur at puberty, predicting and quantifying life-long adaptation, while negating Pauwels' hypothesis that the linea aspera stiffens the femur in bending. This virtual case study of life-long functional adaptation demonstrates the utility of modern computational methods to test mechanobiological hypotheses relevant to evolution of form and function. Further, it suggests a role for the periosteum as a mediator of strong muscular attachments at the linea aspera. Thereafter, measurements of cadaveric periosteum from aged human femora provide unprecedented reference values for clinical translation. They also show a rather surprisingly weak relationship between periosteum's structural properties and prevailing mechanical environment, spawning new research questions regarding the mechanomodulatory processes by which this anisotropic, ‘smart’ material develops and is maintained. Then, building upon the clinical context, a critical sized defect model serves to assess bone regeneration following treatment with a novel surgical membrane designed to replicate periosteum's structural, barrier and cellular functions. High-resolution histomorphometry shows that incorporation of biological factors from autologous periosteum augments bone tissue generation, predominantly via an endochondral ossification pathway. Finally, a mechanistic, paired computational-analytical and multiscale model bridges periosteal mechanics and biology, predicting growth factor-regulated processes of tissue infilling in a simulated defect, and enabling parametric assessment to engineer next-generation periosteal replacements. As a whole, this work advances understanding of periosteal mechanobiology and underscores the need for follow-on studies to address current gaps in knowledge regarding mechanically mediated tissue generation, adaptation and repair.