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 (PO₄3-) 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.
|2011||Russell RGG. Bisphosphonates: the first 40 years. Bone. July 2011;49(1):2-19.|
|2010||Gourion-Arsiquaud S, Allen MR, Burr DB, Vashishth D, Tang SY, Boskey AL. Bisphosphonate treatment modifies canine bone mineral and matrix properties and their heterogeneity. Bone. March 2010;46(3):666-672.|
|2013||Boskey AL. Bone composition: relationship to bone fragility and antiosteoporotic drug effects. BoneKEy Rep. December 2013;2:447.|
|2008||Drake MT, Clarke BL, Khosla S. Bisphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc. September 2008;83(9):1032-1045.|
|2012||Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. June 28, 2012;9(7):676-682.|
|2002||Turner CH. Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int. February 2002;13(2):97-104.|
|2012||Donnelly E, Meredith DS, Nguyen JT, Gladnick BP, Rebolledo BJ, Shaffer AD, Lorich DG, Lane JM, Boskey AL. Reduced cortical bone compositional heterogeneity with bisphosphonate treatment in postmenopausal women with intertrochanteric and subtrochanteric fractures. J Bone Miner Res. March 2012;27(3):672-678.|
|2008||Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone mineralization density distribution in health and disease. Bone. March 2008;42(3):456-466.|
|2011||Yao H, Dao M, Carnelli D, Tai K, Ortiz C. Size-dependent heterogeneity benefits the mechanical performance of bone. J Mech Phys Solids. 2011;59(1):64-74.|
|2007||Tai K, Dao M, Suresh S, Palazoglu A, Ortiz C. Nanoscale heterogeneity promotes energy dissipation in bone. Nat Mater. June 2007;6(6):454-462.|
|2001||Wang X, Bank RA, Tekoppele JM, Agrawal CM. The role of collagen in determining bone mechanical properties. J Orthop Res. 2001;19(6):1021-1026.|
|2014||Wright NC, Looker AC, Saag KG, Curtis JR, Delzell ES, Randall S, Dawson‐Hughes B. The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J Bone Miner Res. November 2014;29(11):2520-2526.|
|2011||Ritchie RO. The conflicts between strength and toughness. Nat Mater. November 2011;10(11):817-822.|
|2008||Campbell GM, Buie HR, Boyd SK. Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT. Osteoporos Int. October 2008;19(10):1409-1419.|
|2005||Boskey AL, DiCarlo E, Paschalis E, West P, Mendelsohn R. Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation. Osteoporos Int. December 2005;16(2):2031-2038.|
|2005||Tai K, Qi HJ, Ortiz C. Effect of mineral content on the nanoindentation properties and nanoscale deformation mechanisms of bovine tibial cortical bone. J Mater Sci Mater Med. October 2005;16(10):947-959.|
|2008||Russell RGG, Watts NB, Ebetino FH, Rogers MJ. Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int. June 2008;19(6):733-759.|
|1997||Paschalis EP, Betts F, DiCarlo E, Mendelsohn R, Boskey AL. FTIR microspectroscopic analysis of normal human cortical and trabecular bone. Calcif Tiss Int. December 1997;61(6):480-486.|
|2016||Demirtas A, Curran E, Ural A. Assessment of the effect of reduced compositional heterogeneity on fracture resistance of human cortical bone using finite element modeling. Bone. October 2016;91:92-101.|
|2014||Cosman F, de Beur SJ, LeBoff MS, Lewiecki EM, Tanner B, Randall S, Lindsay R. Clinician’s guide to prevention and treatment of osteoporosis. Osteoporos Int. October 2014;25(10):2359-2381.|
|2018||Imbert L, Gourion-Arsiquaud S, Villarreal-Ramirez E, Spevak L, Taleb H, van der Meulen MCH, Mendelsohn R, Boskey AL. Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy. PLoS One. September 4, 2018;11(9):e0202833.|
|2006||Viguet-Carrin S, Garnero P, Delmas PD. The role of collagen in bone strength. Osteoporos Int. March 2006;17(3):319-336.|
|2002||Burr DB. The contribution of the organic matrix to bone's material properties. Bone. July 2002;31(1):8-11.|