Bone adapts its structure in response to mechanical loading through tightly regulated cellular and molecular mechanisms. Although osteocytes embedded within the mineralized matrix are widely regarded as the primary mechanosensors in bone, emerging evidence suggests that periosteal cells may also play a critical role in load-induced cortical bone adaptation. The periosteum is essential for cortical bone growth, repair, and regeneration;​ however, the mechanisms governing its mechanical responsiveness remain poorly defined. The overall objective of this thesis is to elucidate the cellular basis and molecular signaling pathways underlying mechanical responses in the periosteum.

In Specific Aim 1, an inducible osteocyte ablation mouse model was used to directly assess the requirement of osteocytes in periosteal mechanoadaptation. Using in vivo uniaxial tibial loading combined with µCT analysis and histological staining, periosteal mechanical responses were evaluated following osteocyte depletion. Despite efficient osteocyte ablation, mechanically induced periosteal bone formation was preserved, demonstrating that periosteal mechanical responses can occur independently of osteocytes and establishing the periosteum as an autonomous mechanosensitive compartment. Furthermore, histological analyses revealed activation and proliferation of periosteal bone surface cells, accompanied by regulated expression of Piezo1, YAP1, and β-catenin following a single loading bout, suggesting that these cells directly sense mechanical cues and mediate periosteal responses.

In Specific Aim 2, lineage-tracing and lineage-ablation mouse models were used to investigate the functional role of periosteal bone surface cells in mechanotransduction. Using the same in vivo tibial loading protocol, along with µCT and histological analyses, periosteal responses to mechanical stimulation were assessed. These periosteal bone surface cells were first identified as PDGFRα⁺ progenitor cells residing on the periosteal bone surface. Following mechanical loading, PDGFRα⁺ periosteal bone surface cells became embedded within the bone matrix and served as the primary source of newly formed osteocytes. Importantly, depletion of these cells markedly reduced periosteal bone formation and abolished load-induced upregulation of Piezo1, YAP1, and β-catenin. These findings demonstrate that PDGFRα⁺ periosteal bone surface cells are essential mechanosensitive mediators of periosteal adaptation.

In Specific Aim 3, single-cell RNA sequencing was employed to characterize early transcriptional responses of periosteal cells following a single bout of mechanical loading. This analysis resolved periosteal cellular heterogeneity and revealed rapid, load-induced gene expression changes in distinct periosteal subpopulations. In periosteal progenitor cells at 4 hours post-loading, differential gene expression analysis identified significant upregulation of mechanosensitive ion channels, including Piezo1 and Mcoln2, as well as osteogenesis-related genes such as Ptgs2 and Ccn1. Gene ontology and pathway analyses revealed enrichment of mechanotransduction, cytoskeletal remodeling, and osteogenic signaling pathways, including PI3K, TNF, EGFR, NF-κB, and JAK-STAT signaling, providing molecular insight into periosteal mechanosensitivity.

Together, the findings of this thesis challenge the prevailing osteocyte-centric model of bone mechanotransduction and identify PDGFRα⁺ periosteal bone surface cells as independent and critical regulators of periosteal bone adaptation. By defining essential periosteal cell populations and their mechanosensitive signaling pathways, this work advances understanding of cortical bone mechanobiology and suggests new therapeutic strategies to enhance bone formation in skeletal disorders such as osteoporosis.