From Genetic Mutation to Skeletal Fragility: Multiscale Profiling of Human Osteogenesis Imperfecta Bone

Abstract

Osteogenesis imperfecta (OI) is a genetically heterogeneous connective tissue disorder characterized by bone fragility, recurrent fractures, skeletal deformities, and substantial variability in clinical severity. Although OI is primarily associated with defects in type I collagen or proteins involved in its processing, the mechanisms through which genetic abnormalities translate into heterogeneous bone phenotypes remain incompletely understood. This thesis investigated OI bone across multiple biological scales, integrating collagen biochemistry, histopathology, molecular characterization, single-cell transcriptomics, and evidence on emerging anabolic therapy. Biochemical analysis of pediatric cortical bone demonstrated markedly reduced collagen content per dry bone weight in OI and increased lysyl hydroxylation. The degree of lysyl hydroxylation was related to mutation position and substitution type, supporting delayed triple-helix folding as an important determinant of collagen overmodification. Histological and imaging evidence further showed that OI is characterized by abnormalities extending beyond collagen composition, including reduced cortical thickness, disturbed lamellar organization, persistence of woven bone, altered mineralization, increased osteocyte lacunar density, and intracortical porosity. Importantly, several genetic subtypes exhibited characteristic histological patterns. Integrated histological, polarized-light microscopic, and Raman spectroscopic analysis of human cortical bone confirmed disrupted lamellar formation, abnormal collagen organization, increased osteocyte lacunar area, and alterations in mineral- and matrix-related molecular signals. These abnormalities were generally more pronounced in severe OI subtypes. Subtype-associated features included a fish-scale lamellar pattern in OI type VI and extensive woven bone in severe forms such as types VIII and XIV. Single-cell RNA sequencing of human bone and bone marrow further demonstrated that OI affects the cellular microenvironment beyond collagen-producing cells. Mesenchymal stromal cell proportions were reduced, with downregulation of osteogenesis-related genes including RUNX1 and BMP5. Changes in osteoblast, osteoclast-lineage, monocyte, and other immune populations differed between OI subtypes, suggesting subtype-dependent disturbances in bone formation, resorption, and immune–skeletal interactions. Finally, a systematic review of anti-sclerostin therapy showed that sclerostin inhibition can substantially improve bone mass, cortical and trabecular architecture, and whole-bone strength. However, improvements in intrinsic tissue-level material properties and matrix quality were less consistent, particularly in severe OI, indicating that increasing bone quantity does not necessarily normalize bone quality. Together, these findings establish OI as a multiscale disorder in which genetic defects interact with collagen matrix abnormalities, disrupted tissue organization, and altered cellular remodeling environments to determine skeletal fragility. A multiscale, genotype-informed approach may therefore improve phenotypic stratification and support more personalized therapeutic strategies targeting both bone quantity and bone quality.