## 3 Bone Pathologies: Alterations to Bone Ultrastruct and Mechanics
### Osteogenesis Imperfecta
(**a**) Ultrastruct changes: (**i**) TEM images showing (1) a single collagen fibril and (1–3) its intrafibrillar crystals in a type IV OI patient bone. The distribution of the longitudinal angle \(\alpha\) of  
the crystals is plotted in (4). For reference, corresponding data for healthy bone from [150] are plotted. Figures reprinted from [146]. **(ii)** Altered fibril morphology in type III OI patient bone. Figure reprinted from [147], with permission from [PERSON]. **(iii)** TEM images comparing the collagen fibril organizations in WT and OIM bone. Figures reprinted from [32], with permission from the American Chemical Society. **(b)** Chemical changes: **(i)** Shifted bone mineral density distribution (BMDD) curves, indicating slight hypermineralization compared to healthy human bone. Figure is replotted using data from [136]. **(ii)** Nanoscale changes in the carbonate content of minerals in OIM bone. Spectra reprinted from [32], with permission from the American Chemical Society. For detailed spectral interpretation and peak assignment (labeled A-E), readers are referred to the original paper [32]. **(c)** Micromechanical properties of healthy vs. OI bone under compression using micropillar samples. Compressive strength of bone was found to increase with OI severity. Figures reprinted with permission from [46]. Chemical alterations in OI-affected bone also start at the nanoscale. Biochemically, dysregulation of NCP expressions is common in OI and varies with disease severity: with reduced levels of ON (SPARC) and increased BSP content [151]. As a result, NCP-collagen interactions become compromised in recessive OI. The bone minerals in OI pathology were less crystalline, and reductions in both the carbonate and phosphate contents have been recorded (Figure 5(bi)) [142; 152; 39]. However, these values were averaged across the entire specimen examined, and localized alterations could not be identified. [PERSON] et al. employed spatially-resolved STEM-EELS to probe the fine structures of carbon K and nitrogen K edges in the high-energy loss region of the spectrum, revealing nanoscale chemical changes in OIM bone tissue [32]. In Figure 5(bii), the peak-labeled D, with an energy loss of 290.0-290.2 eV, corresponds to the transitions of carbonate groups, and the shoulder E is indicative of carbonate 1s \(\rightarrow\)\(\sigma\)*-C and \(\pi\)*-C transitions [32]. Using this spectral information, the authors identified highly heterogeneous distributions of carbonate-rich and carbonate-deficient regions in OI bone compared to WT control [32]. This alludes to a defective mineralization process, with a highly localized and spatially inhomogeneous molecular origin that calls for further examination. Overall, ultrastructural defects in both the mineral and collagen phases generally correlate well with the severity of the OI type [147]. Numerous studies have reported modified whole-bone-level mechanics of OI bone, including decreased compressive elastic modulus, reduced bending strength, increased compressive strength, increased hardness, and, most notably, heightened brittleness compared to healthy counterparts [142; 152; 153; 143]. For smaller length scales, an increased compressive modulus was found in the extracellular matrix of OI bone compared to WT using FIB-machined micropillar samples, and the magnitude of increase appeared to correlate with the degree of bone mineralization and disease severity (Figure 5c) [46]. In another recent study, by subjecting OIM bone to ex-situ micropillar compression tests and through finite element modeling, [PERSON] et al. proposed the possible mechanical behavior of OI bone at the fibrillar level [153]. Under compressive stress, OIM bone was found to fail in a manner consistent with brittle materials: the machined bone micropillars cracked and underwent splitting in the longitudinal direction, whereas the WT control after mechanical failure displayed a shear failure plane with localized plastic deformation zones [153].