Nano- to microscale chemical heterogeneity plays a role in fracture prevention for osteoporotic bone. Once significant bone loss has occurred, the ability to restore biomechanical function may differ based on the drug chosen. In this study, we seek to understand changes in chemical composition linked to disease and treatment in lumbar vertebral trabecular bone using a treatment animal model. To the best of our knowledge, this is the first published treatment animal model to understand osteoporosis.

It should be noted that Merck, Inc. designed and performed all experiments relating to the treatment animal model including μCT, mechanical testing and BMD determination. Study results and sliced LV samples were provided to our group at the University of Michigan for analysis and publication.

To assess this question, bisphosphonate (alendronate, ALN), cathepsin K inhibitor (MK-0674, CatKi), and denosumab (DMab) were employed in treatment mode to compare the relative changes to trabecular bone in ovariectomized (OVX) cynomolgus monkeys. Lumbar vertebrae (LV) bone mineral density (BMD) values taken two years post-surgery prior to drug treatment show a 10-15% decrease (p < 0.05) for all OVX animals. OVX animals were then treated with vehicle (VEH), ALN (0.03 mg/kg weekly), CatKi MK-0674 (0.6 or 2.5 mg/kg daily), and DMab (2.5 mg/kg weekly subcutaneously) for two years and compared to a Sham surgery group.

Ex-vivo microcomputed tomography (μCT) of LV2 and compression testing of LV4-6 were used to measure trabecular bone microstructure and changes in bone mechanics, respectively. After two years of treatment, ALN-treated animals showed no significant difference in μCT or biomechanical parameters when compared to Veh. However, treatment with CatKi resulted in a 30% increase in yield and peak loads, and apparent peak and yield stress as compared to Veh (p < 0.08) and gave average mechanical values greater than the Sham. Intriguingly, these changes were realized despite no significant differences in mean values of trabecular bone morphologic parameters.

To understand changes in structure and composition, atomic force microscopy - infrared spectroscopy (AFM-IR) was used to obtain topographical information coupled to chemical composition/structure for cross-sections of lumbar vertebrae 4 (LV4). The average unraveled collagen content, average organic matrix, mineral content, and acid phosphate (PO₄^3-) substitution for each group was evaluated.

Average unraveled to intact (1732 - 1748 cm^-1/1656 - 1672 cm^-1) collagen integrated peak ratios were obtained for Sham, OVX, ALN, CatKi with DMab being significantly different from all other groups. From these results, CatKi is similar to Sham, ALN is similar to OVX and DMab is completely different from all other groups. Average mineral to matrix (900-1200/1500-1800 cm^-1) ratios obtained for Sham, OVX, ALN, CatKi and DMab groups. No significant difference was detected. Average acid phosphate substitution (1128/ 1096 cm ^-1 intensity ratio) obtained for Sham, OVX, ALN, CatKi and DMab groups. CatKi was significantly different (p < 0.05) from OVX, ALN and DMab, yet no significant difference was detected for Sham. CatKi shows a preservation, or reversal, to normal levels of acid phosphate substitution. Unraveled collagen heterogeneity that appears as a function of OVX could be chemical biomarker of increased bone turnover. With nano-scale resolution, we are capable of detecting compositional changes smaller than those detected with conventional spectroscopic techniques.