## 3 Bone Pathologies: Alterations to Bone Ultrastruct and Mechanics
### Osteoporosis
Starting at the molecular scale, tropocollagen molecules that self-assemble into collagen fibrils and subsequently into collagen fibers can be intrinsically defective as a result of defects in the \(COLIA1\) gene that encodes for the \(\alpha 1\) type I collagen, where the gene is associated with osteoporosis in man [132]. Early work on biochemical analysis revealed a reduction in the quantity of Type I collagen, as well as decreased amount of total collagen crosslinks in idiopathic osteoporotic bone compared to healthy bone [133]. An overall elevated collagen maturity was also detected due to an increased level of mature pyridinoline crosslinking and a reduction in immature dihydroxylysino-norleucine (DHLNL) crosslinking [133], where the degree of such enzymatic crosslinking directly impacts fibril mechanics [134]. In both senile and ovariectomy-induced (postmenopausal) mice, irregularities in collagen fibril morphologies were observed: the collagen fibrils were, on average, shorter and possessed a reduced diameter compared to normal counterparts (Figure 4(aiiii)) [130; 133; 135]. The cross-sectional TEM image of the collagen fibrils shown in Figure 4 also revealed a loss of their characteristic quasi-hexagonal packing, leading to a disordered arrangement in the transverse direction. In the longitudinal direction, the fib rillar texture was also more random in osteopororotic bone (Figure 4(aiv)) [130; 133; 135]. This was further validated using spatially-resolved SAXS maps in a mouse model with excessive endogenous steroids [129]. On average, the mineral-to-matrix ratio is lower in osteopororotic bone than in normal bone (Figure 4(bii)), with bone mineral density distribution shifting to lower values, indicating a hypomineralized bone (Figure 4(bi)). Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been used to distinguish between local chemical features in normal and osteopororotic bone [44]. Compared to the typical spatial resolution of 1-10 \(\upmu\)m for vibrational spectroscopic imaging (e.g., FTIR), the lateral resolution can reach less than 300 nm for ToF-SIMS imaging, elucidating sub-\(\upmu\)m scale variations in chemistry. Figure 4: Altered bone ultrastructure, chemistry, and nanomechanics in _osteoporosis_ pathology. (**a**) Ultrastructual changes: (**i**) high-resolution TEM images and SAED patterns of the cortical bone  
from sham/WT mice and ovariectomized postmenopausal osteeoporotic mice, showing differences in the packing arrangement of the mineral nanocrystals. Figures reprinted from [130], with permission from the American Chemical Society. (**ii**) Superimposed X-ray intensity contour map and quiver plot showing mineral crystal orientation in WT vs. steroid-induced osteopororetic bone. Figures reprinted from [129], with permission from Elsevier. (**iii**) TEM images of collagen fibrils and the corresponding histograms for collagen fibril diameter. Figures reprinted from [133], with permission from Taylor & Francis. (**iv**) TEM images of collagen fibrillar organizations in healthy vs. osteopororetic rat bone. Figures reprinted from [135], with permission from Elsevier. A key is provided to help differentiate between different types of osteoporosis (idiopathic/general, senile, postmenopausal, and steroid-induced) and between human and animal (mainly mice or rats) samples. (**b**) Chemical changes: (**i**) Shifted bone mineral density distribution (BMDD) curves, indicating hypomineralization compared to healthy human bone. Figure is replotted using data from [136]. FTIR micro-spectroscopic images showing spatially resolved mineral (**ii**) crystallinity and (**iii**) mineral-to-matrix ratio, respectively, for healthy and osteopororetic patient bone. Figures reprinted from [41]. (**c**) Altered in situ deformation behavior in WT and ovariectomized mice using nanopillar samples: (1, 4) AFM images, (2, 5) SEM images, and (3, 6) TEM images of a crack. Figures reprinted from [130], with permission from the American Chemical Society.