The periosteum serves as a stabilizing boundary membrane to the bone it envelopes and contains mechanosensitive progenitor cells capable of promoting new bone formation. Knowledge of the structure-function relationships underlying the dynamic mechanical and regenerative properties of the periosteum is lacking. The following work investigates the structure-function relationships present in periosteum at both the tissue and cellular level. At the tissue level, periosteum permeability is characterized and found to exhibit barrier membrane properties. Subsequently, cell scale studies investigate the molecular characteristics of periosteum cells responsible for enabling tissue-level barrier membrane properties. Lastly, a biomimetic membrane system for investigating cell-cell adhesions of periosteum cells is developed. Culture of periosteum cells on the model membrane system results in signs of early osteogenic lineage commitment. Characterization of structure-function relationships in periosteum across multiple lengths scales provides knowledge valuable for the development of predictive computational models and tissue engineered periosteal replacement membranes.