In Vivo Response of Type I Diabetic Bone to Mechanical Loading Depends on gender and/or Disease Severity

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1 In Vivo Response of Type I Diabetic Bone to Mechanical Loading Depends on gender and/or Disease Severity Ashutosh Parajuli 1, Xiaoyu D. Gu 1, Xiaohan Lai 1, Hong Zhang 2, Mia M. Thi, PhD 3, Christopher Price, Ph.D. 1, Liyun Wang, Ph.D University of Delaware, Newark, DE, USA, 2 Rowan University, Glassboro, NJ, USA, 3 Albert Einstein College of Medicine, Bronx, NY, USA. Disclosures: A. Parajuli: None. X.D. Gu: None. X. Lai: None. H. Zhang: None. M.M. Thi: None. C. Price: None. L. Wang: None. Introduction: Diabetic patients showed increased risk of fracture and bone loss [1], possibly due to impaired osteoblast bone formation [2,3], compromised bone structure [4], increased cortical porosity [5], inferior mechanical properties [6], and impaired osteocyte mechanosensing [7,8]. Since some pharmaceutical treatments such as thiazolidinediones (TZDs), the insulin-sensitizing PPARy agonists, are found to have negative effects on bone quality [9], non-pharmacological interventions such as mechanical stimulation (one of the most potent anabolic stimuli for bone) could be used as supplemental or alternative treatments. However, the response of diabetic bone to mechanical loading in vivo is not fully understood. The objective of this study was to examine bone structural and histomorphometrical changes in diabetic and normal mice after short-term unilateral ulnar loading. Methods: The right ulnae of 29-week old male and female heterozygous C57BL/6-Ins2 Akita (Akita) and wild type C57BL/6J (WT) mice (Jackson Laboratory; n=5-7 mice/group) were subjected to cyclic axial loading to induce ~3500 microstrain on the lateral mid-shaft surface. Strain gauging [10] was performed in a separate set of animals (n>2 ulnae/group) to determine the peak load for each group, which was found to be 2.7N for Akita females (n=7), 3N for WT females (n=5), 2.2N for Akita males (n=5) and 3N for WT males. The loading was applied at 2Hz, 3 min/day, and for 5 consequent days as published previously [10]. The mice received calcein labels (10mg/kg) on Day 4 and Day 15 and sacrificed on Day 18 since the beginning of the loading. Both loaded and contralateral non-loaded ulnae were harvested for structural and dynamic histomorphometry analysis using established protocols [10]. Fasting blood glucose level was determined using an OneTouch glucometer and retro-orbital blood either before or after the 5 loading sessions. All data are presented as mean±standard deviation. Paired Student s t-tests were used to compare responses between loaded and non-loaded ulnae within same animals; and un-paired Student s t-tests were used for comparisons between WT and Akita diabetic groups. Significance was set at p<0.05. Results: Females: Akita female mice showed no significant difference in body weight ( vs gm), but an elevated fasting blood glucose level ( vs mg/dl, p=0.002) compared with those of WT females. After five days of mechanical loading, WT females demonstrated robust bone formation in terms of increased cortical bone area (Ct.B.Ar, +15.6%, p=0.03) and cortical thickness (Ct.Th, +9.9%, p=0.005), increased periosteal mineralizing surface (Ps.MS/BS, %, p=0.004), mineral apposition rate (Ps.MAR, p=0.01), and bone formation rate (Ps.BFR/BS, p=0.02) in loaded ulnae compared with non-loaded ulnae. Akita females also showed robust bone formation in loaded ulnae in terms of Ct.B.Ar (+6.5%, p=0.02), Ct.Th (+6.5%, p=0.005) and increased Ps.MS/BS, Ps.MAR, and Ps. BFR/BS (p<0.05). Both WT and Akita females showed increased mineralizing surface in endosteal surface (Ec.MS/BS, p<0.05). When comparing the relative changes (loaded minus non-loaded indices), the WT and Akita females did not differ significantly in the structural changes (Δ Ct.B.Ar, Fig. 1A), and the periosteal and endosteal bone label indices (Fig. 2), except that rec.ms/bs in Akita females was even higher. Males: Akita males, however, showed more severe effects of diabetes with a -28.5% decrease in body weight ( vs gm, p<0.0001) and a highly elevated fasting blood glucose level ( vs mg/dl, p<0.0001). As expected, WT males showed robust bone formation after loading with increased Ct.B.Ar (+13.6%, p<0.001) and Ct.Th (+11.6%, p<0.001), increased Ps.MS/BS (+19.9%, p=0.01), increased Ps.MAR (p=0.04), and increased Ec.Ms/BS (+12.3%, p=0.03) in loaded ulnae compared with non-loaded ulnae. In contrast, Akita males did not show anabolic responses in most of the histomorphometry indices, except for Ps.MS/BS (+119.3%, p=0.01) and Ec.MAR (p=0.04). When comparing the relative changes in bone structure and bone labeling, the Akita males demonstrated much attenuated responses to loading when compared with WT controls (Fig. 1B, Fig. 3). Discussion: We found that diabetic bone s response to mechanical loading depends on gender and/or the severity of diabetes. As expected, WT males and females both responded to the current loading regimen (~3500micronstrain, 2Hz, 3 min/day for 5 days) with robust bone formation in terms of the structural (Fig. 1) and dynamic bone label parameters (Figs. 2 and 3). Under the same mechanical stimulation, female Akita mice responded as well as the WT controls. It is noted that Akita females, despite elevated fasting blood glucose levels (+44%), maintained normal body weights. In contrast, the Akita males had extremely higher glucose levels (+227%) and significantly lower body weights (-28.5%) than WT males. The more severe diabetic phenotype in the Akita males may account for their attenuated responses to mechanical stimulation. It is not clear whether female hormones such as estrogen and/or the milder diabetic phenotype improved the response of female Akita mice to loading. Further studies are needed to elucidate this gender-dependent response and to study the long-term effects of loading on both trabecular and

2 cortical bone structure and mechanical properties. Overall, the current studies suggest that diabetic bone can respond to mechanical loading under certain conditions and that mechanical interventions may be useful to prevent the deterioration of bone properties and reduce fracture risk in diabetic patients. Significance: Diabetes and diabetic related bone fractures affects millions of patients, resulting in a significant socioeconomic burden and negatively impacting patients quality of life. Non-pharmaceutical interventions (including exercise) that could rescue the weak bone phenotypes in diabetic patients are safe and economical and could be used as an alternative therapy approach. Acknowledgments: This study was supported by NIH AR and P30GM References: [1] Vestergaard (2007) Osteoporos Int 18:427; [2] Kiel (2007) Bone. 40:587; [3] Santos (2009) JOR 27:1280; [4] Melton (2008) J Clin Endocrinol Metab 93:4804; [5] Patsch (2013) JBMR 28:313; [6] Burghardt (2010) J Clin Endocrinol Metak 95:5045; [7] Liu 2013 ORS abstract; [8] Li 2013 ORS abstract; [9]Loke (2009) CMAJ 180:32; [10] Li (2005), J Biol Chem 30:280.

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4 ORS 2014 Annual Meeting Poster No: 0598

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