Browsing by Author "Hammond, Max A."
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Item Calcitriol suppression of parathyroid hormone fails to improve skeletal properties in an animal model of chronic kidney disease(Karger, 2016) Newman, Christopher L.; Tian, Nannan; Hammond, Max A.; Wallace, Joseph M.; Brown, Drew M.; Chen, Neal X.; Moe, Sharon M.; Allen, Matthew R.; Department of Anatomy & Cell Biology, IU School of MedicineBACKGROUND: Chronic kidney disease (CKD) leads to complex metabolic changes and an increased risk of fracture. Currently, calcitriol is the standard of care as it effectively suppresses parathyroid hormone (PTH) levels in CKD patients. While calcitriol and its analogs improve BMD and reduce fractures in the general population, the extension of these benefits to patients with advanced kidney disease is unclear. Here, the impact of calcitriol on the skeleton was examined in the setting of reduction in PTH. METHODS: Male Cy/+ rats, a PKD-like CKD model, were treated with either vehicle or calcitriol for 5 weeks. Their normal littermates served as controls. Animals were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole bone mechanics and bone quality). RESULTS: PTH levels were significantly higher (12-fold) in animals with CKD compared to normal controls. CKD animals also exhibited negative changes in bone structural and mechanical properties. Calcitriol treatment resulted in a 60% suppression of PTH levels in animals with CKD. Despite these changes, it had no impact on bone volume (cortical or cancellous), bone turnover, osteoclast number or whole bone mechanical properties. CONCLUSIONS: These data indicate that while calcitriol effectively lowered PTH in rats with CKD, it did little to prevent the negative effects of secondary hyperparathyroidism on the skeleton.Item Cortical Bone Mechanical Properties Are Altered in an Animal Model of Progressive Chronic Kidney Disease(2014-06) Newman, Christopher L.; Moe, Sharon M.; Chen, Neal X.; Hammond, Max A.; Wallace, Joseph M.; Nyman, Jeffry S.; Allen, Matthew R.Chronic kidney disease (CKD), which leads tocortical bone loss and increasedporosity,increases therisk of fracture. Animal models have confirmed that these changes compromise whole bone mechanical properties. Estimates from whole bone testing suggest that material properties are negatively affected, though tissue-level assessmentshavenot been conducted. Therefore, the goal of the present study was to examine changes in cortical bone at different length scales using a rat model with theprogressive development of CKD. At 30 weeks of age (~75% reduction in kidney function), skeletally mature male Cy/+ rats were compared to their normal littermates. Cortical bone material propertieswere assessed with reference point indentation (RPI), atomic force microscopy (AFM), Raman spectroscopy,and high performance liquid chromatography (HPLC). Bones from animals with CKD had higher (+18%) indentation distance increase and first cycle energy dissipation (+8%) as measured by RPI.AFM indentation revealed a broader distribution of elastic modulus values in CKD animals witha greater proportion of both higher and lower modulus values compared to normal controls. Yet, tissue composition, collagen morphology, and collagen cross-linking fail to account for these differences. Though the specific skeletal tissue alterations responsible for these mechanical differences remain unclear, these results indicate that cortical bone material properties are altered in these animals and may contribute to the increased fracture risk associated with CKD.Item Differential effects of Epigallocatechin-3-gallate containing supplements on correcting skeletal defects in a Down syndrome mouse model(Wiley Blackwell (John Wiley & Sons), 2016-04) Abeysekera, Irushi; Thomas, Jared; Georgiadis, Taxiarchis M.; Berman, Alycia G.; Hammond, Max A.; Dria, Karl J.; Wallace, Joseph M.; Roper, Randall J.; Department of Biology, School of ScienceSCOPE: Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is characterized by a spectrum of phenotypes including skeletal abnormalities. The Ts65Dn DS mouse model exhibits similar skeletal phenotypes as humans with DS. DYRK1A, a kinase encoded on Hsa21, has been linked to deficiencies in bone homeostasis in DS mice and individuals with DS. Treatment with Epigallocatechin-3-gallate (EGCG), a known inhibitor of Dyrk1a, improves some skeletal abnormalities associated with DS in mice. EGCG supplements are widely available but the effectiveness of different EGCG-containing supplements has not been well studied. METHODS AND RESULTS: Six commercially available supplements containing EGCG were analyzed, and two of these supplements were compared with pure EGCG for their impact on skeletal deficits in a DS mouse model. The results demonstrate differential effects of commercial supplements on correcting skeletal abnormalities in Ts65Dn mice. Different EGCG-containing supplements display differences in degradation, polyphenol content, and effects on trisomic bone. CONCLUSION: This work suggests that the dose of EGCG and composition of EGCG-containing supplements may be important in correcting skeletal deficits associated with DS. Careful analyses of these parameters may lead to a better understanding of how to improve skeletal and other deficits that impair individuals with DS.Item Differential effects of Epigallocatechin-3-gallate containing supplements on correcting skeletal defects in a Down syndrome mouse model(Wiley, 2016-04) Abeysekera, Irushi; Thomas, Jared; Georgiadis, Taxiarchis M.; Berman, Alycia G.; Hammond, Max A.; Dria, Karl J.; Wallace, Joseph M.; Roper, Randall J.; Biology, School of ScienceSCOPE: Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is characterized by a spectrum of phenotypes including skeletal abnormalities. The Ts65Dn DS mouse model exhibits similar skeletal phenotypes as humans with DS. DYRK1A, a kinase encoded on Hsa21, has been linked to deficiencies in bone homeostasis in DS mice and individuals with DS. Treatment with Epigallocatechin-3-gallate (EGCG), a known inhibitor of Dyrk1a, improves some skeletal abnormalities associated with DS in mice. EGCG supplements are widely available but the effectiveness of different EGCG-containing supplements has not been well studied. METHODS AND RESULTS: Six commercially available supplements containing EGCG were analyzed, and two of these supplements were compared with pure EGCG for their impact on skeletal deficits in a DS mouse model. The results demonstrate differential effects of commercial supplements on correcting skeletal abnormalities in Ts65Dn mice. Different EGCG-containing supplements display differences in degradation, polyphenol content, and effects on trisomic bone. CONCLUSION: This work suggests that the dose of EGCG and composition of EGCG-containing supplements may be important in correcting skeletal deficits associated with DS. Careful analyses of these parameters may lead to a better understanding of how to improve skeletal and other deficits that impair individuals with DS.Item Even With Rehydration, Preservation in Ethanol Influences the Mechanical Properties of Bone and How Bone Responds to Experimental Manipulation(Elsevier, 2017-04) Vesper, Evan O.; Hammond, Max A.; Allen, Matthew R.; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyTypically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0M or 0.6M ribose for 1week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (-16.3%), stiffness (+24.5%), strain to yield (-13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%-68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation).Item Hysteroscopic Sterilization Device Follow-Up Rate: Hysterosalpingogram Versus Transvaginal Ultrasound(Elsevier, 2018) Jeirath, Neha; Basinski, Cindy M.; Hammond, Max A.; Obstetrics and Gynecology, School of MedicineStudy Objective To assess if follow-up confirmation testing 3 months after hysteroscopic sterilization with the Essure device improved with recommendation for transvaginal ultrasound (TVUS) versus hysterosalpingogram (HSG). Design Retrospective, observational case-controlled cohort study (Canadian Task Force classification II-2) Setting Two physician private practice in Evansville, Indiana Patients Compliance rates for a TVUS confirmation test on 100 women who underwent hysteroscopic sterilization compared to a previously published cohort of 1004 women who were scheduled to undergo HSG confirmation test. Intervention Acquisition of 3 month confirmation testing after Essure hysteroscopic sterilization Measurement and Main Results All women who underwent Essure hysteroscopic sterilization with recommendation for TVUS confirmation testing between July 2015 and January 2017 were compared to a previously published cohort of 1004 patients with recommendation for HSG confirmation testing (HSG cohort). In addition, an HSG subgroup cohort (HSG subgroup) similar in size and closest chronology to the TVUS cohort was drawn from the original 1004 patients and analyzed for HSG follow-up. Records for all patients were reviewed for demographic, procedural, confirmation testing, and outcome data. One hundred patients were identified with successful Essure device placement and a recommendation for TVUS confirmation testing. Eighty-eight (88.0%) patients returned for TVUS at 3 months. In the HSG cohort, 1004 successful Essure devices were placed and 778 patients returned for the recommended HSG follow-up (77.5%). There was a significantly higher follow-up rate for TVUS compared to the HSG cohort (88.0% vs 77.5%, p = 0.008). In the HSG subgroup, 184 patients were identified and 133 patients presented for HSG follow-up (72.3%) indicating a significantly higher follow-up rate in the TVUS cohort (88.0% vs 72.3%, p = 0.001). No pregnancies after any confirmation testing were noted. Conclusion Confirmation testing with transvaginal ultrasound rather than hysterosalpingogram 3 months after Essure device placement results in increased patient compliance that may lead to improved patient outcomes with reduction of unintended pregnancy.Item Incorporating tissue anisotropy and heterogeneity in finite element models of trabecular bone altered predicted local stress distributions(Springer, 2018-04) Hammond, Max A.; Wallace, Joseph M.; Allen, Matthew R.; Siegmund, Thomas; Anatomy and Cell Biology, School of MedicineTrabecular bone is composed of organized mineralized collagen fibrils, which results in heterogeneous and anisotropic mechanical properties at the tissue level. Recently, biomechanical models computing stresses and strains in trabecular bone have indicated a significant effect of tissue heterogeneity on predicted stresses and strains. However, the effect of the tissue-level mechanical anisotropy on the trabecular bone biomechanical response is unknown. Here, a computational method was established to automatically impose physiologically relevant orientation inherent in trabecular bone tissue on a trabecular bone microscale finite element model. Spatially varying tissue-level anisotropic elastic properties were then applied according to the bone mineral density and the local tissue orientation. The model was used to test the hypothesis that anisotropy in both homogeneous and heterogeneous models alters the predicted distribution of stress invariants. Linear elastic finite element computations were performed on a 3 mm cube model isolated from a microcomputed tomography scan of human trabecular bone from the distal femur. Hydrostatic stress and von Mises equivalent stress were recorded at every element, and the distributions of these values were analyzed. Anisotropy reduced the range of hydrostatic stress in both tension and compression more strongly than the associated increase in von Mises equivalent stress. The effect of anisotropy was independent of the spatial redistribution high compressive stresses due to tissue elastic heterogeneity. Tissue anisotropy and heterogeneity are likely important mechanisms to protect bone from failure and should be included for stress analyses in trabecular bone.Item Multi-scale Analysis of Bone Chemistry, Morphology and Mechanics in the oim Model of Osteogenesis Imperfecta(2014-08) Bart, Zachary R.; Hammond, Max A.; Wallace, Joseph M.Osteogenesis imperfecta is a congenital disease commonly characterized by brittle bones and caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The oim model has a natural collagen mutation, converting its heterotrimeric structure (two α1 and one α2 chains) into α1 homotrimers. This mutation in collagen may impact formation of the mineral, creating a brittle bone phenotype in animals. Femurs from male wild type (WT) and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanoscale that may partially contribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure obtained from µ-Computed Tomography and Raman spectroscopy indicate a smaller bone with reduced trabecular architecture and altered chemical composition. Decreased tissue material properties in oim/oim mice are likely driven by changes in collagen fibril structure, decreasing space available for mineral nucleation and growth, as supported by a reduction in mineral crystallinity. Multi-scale analyses of this nature offer much in assessing how molecular changes compound to create a degraded, brittle bone phenotype.Item Nanoscale Changes in Collagen are Reflected in Physical and Mechanical Properties of Bone at the Microscale in Diabetic Rats(2014-03) Hammond, Max A.; Gallant, Maxime A.; Burr, David B.; Wallace, Joseph M.Diabetes detrimentally affects the musculoskeletal system by stiffening the collagen matrix due to increased advanced glycation end products (AGEs). In this study, tibiae and tendon from Zucker diabetic Sprague–Dawley (ZDSD) rats were compared to Sprague–Dawley derived controls (CD) using Atomic Force Microscopy. ZDSD and CD tibiae were compared using Raman Spectroscopy and Reference Point Indentation (RPI). ZDSD bone had a significantly different distribution of collagen D-spacing than CD (p = 0.015; ZDSD n = 294 fibrils; CD n = 274 fibrils) which was more variable and shifted to higher values. This shift between ZDSD and CD D-spacing distribution was more pronounced in tendon (p < 0.001; ZDSD n = 350; CD n = 371). Raman revealed significant increases in measures of bone matrix mineralization in ZDSD (PO43 − ν1/Amide I p = 0.008; PO43 − ν1/CH2 wag p = 0.047; n = 5 per group) despite lower bone mineral density (aBMD) and ash fraction indicating diabetes may preferentially reduce the Raman signature of collagen. Decreased indentation distance increase (p = 0.010) and creep indentation distance (p = 0.040) measured by RPI (n = 9 per group) in ZDSD rats suggest a matrix more resistant to indentation under the high stresses associated with RPI at this length scale. There were significant correlations between Raman and RPI measurements in the ZDSD population (n = 18 locations) but not the CD population (n = 16 locations) indicating that while RPI is relatively unaffected by biological noise, it is sensitive to disease-induced compositional changes. In conclusion, diabetes in the ZDSD rat causes changes to the nanoscale morphology of collagen that result in compositional and mechanical effects in bone at the microscale.Item The Phosphate/Amide I ratio is Reduced by in vitro Glycation and may Correlate with Fracture Toughness(Office of the Vice Chancellor for Research, 2015-04-17) Hammond, Max A.; Berman, Alycia G.; Wallace, Joseph M.Introduction: Advanced glycation end products (AGEs) form when reducing sugars react with proteins. In bone AGEs can form in type I collagen which results in non-enzymatically derived crosslinks. While enzymatic crosslinks play an important role in strengthening the collagen matrix, non-enzymatic crosslinks are believed to reduce toughness. AGEs accumulate in bone over time and play an important role in reducing bone quality particularly in aging and diabetic patients who accumulate AGEs more rapidly due to increases in circulating glucose. Non-enzymatic glycation of bone can be modeled experimentally by soaking samples in a sugar solution which allows decades worth of AGE accumulation to occur in a short time. AGEs are primarily measured using fluorescence measurements or high performance liquid chromatography (HPLC). Spectroscopic techniques have been developed to determine enzymatic crosslinking maturity by detecting perturbations in collagen structure in the Amide I region and it may be possible to detect similar changes caused by AGEs. We hypothesized that the formation of AGEs in collagen would perturb the Amide I band of Raman spectra causing changes to the mineral to matrix ratio (MMR) which would correlate with AGE-induced mechanical changes in an in vitro ribose soaking experiment. If changes due to non-enzymatic glycation can be detected in the Amide I band, Raman spectroscopic techniques could be developed to assess the presence of AGEs in a non-destructive and widely available manner. Methods: Five femurs were harvested from male hounds from a previous IACUC approved study. From the mid-diaphysis, six beams ~1.4 x 4 x 24 mm were sectioned from each bone. Two beams from each sample were randomly assigned to one of three groups. One of those beams was sanded to 1.4 x 2 x 20 mm for fracture toughness testing while the other was used for Raman spectroscopy and Reference Point Indentation (RPI). All beams were soaked for 14 consecutive days at 37°C in solutions containing 1% Pen-Strep, 1.3mM CaCl2 and either no ribose (Control), 0.2M ribose (Low), or 0.6M ribose (High) in Hank’s Balanced Salt Solution with solutions changed every other day. After soaking, a notch was started in the sanded beam with a diamond wire sectioning saw and then sharpened by hand with a razor using a 1μm diamond suspension. Notched beams were submerged in fluid and loaded in displacement control to 0.03mm, unloaded to 0.015mm, held for 10s, then cycled until failure with a 0.05mm load, a 0.02 unload, and a 10s hold. J-R curves were calculated using ASTM E1820-5a to obtain initiation stress intensity factor (KIc) and maximum stress intensity factor (Kmax). Raman spectra were acquired at five points along the length of the second beam using a LabRAM HR 800 with a 660nm laser focused to a spot size of ~10μm. After baseline correction, OriginPro 8.6 was used to calculate MMR as the area of the PO43- ν1 peak over the area of the Amide I band. Following Raman spectroscopy, co-localized RPI was performed at each Raman location using 10 cycles of a 5N force at 2Hz. One-way ANOVA tested mean differences between samples. Pearson product-moment correlation coefficients were calculated between MMR and parameters from RPI and fracture toughness. All values are presented as mean ± standard deviation and all statistics were carried out using SAS 9.4. Results: Raman spectroscopy and RPI were not performed on one sample from the Low group. Data were not available for one Control sample and Kmax was excluded for one High sample. Neither KIc nor Kmax were significantly different between groups (Control: 6.59 ± 0.42, 13.55 ± 1.38 MPa√m; Low: 6.19 ± 1.98, 14.80 ± 2.00 MPa√m; High: 6.84 ± 1.18, 15.25 ± 2.35 MPa√m). MMR was significantly different between groups (p=0.039). Tukey HSD post-hoc tests revealed that Control (2.45 ± 0.37) was significantly greater than High (1.85 ± 0.20) while Low was intermediate (2.18 ± 0.37) but not significantly different. No significant differences were observed with RPI. A weak positive correlation was observed between average creep indentation increase (CID) and MMR (R2=0.079, p=0.0185) but no other RPI measurements were correlated with MMR. Two influential points, determined by a Cook’s distance > 4/n, were excluded from the regression KIc to MMR. A mild trend was observed between KIc and MMR but the fit did not reach significance (R2=0.334, p=0.0628). Discussion: Because samples were all from the same 5 animals and randomized into groups, any differences between groups arose from the soaking in solutions of different concentrations of ribose. AGEs were not measured to confirm the expected dose-dependent increase, but noticeable browning occurred in the High group which was less pronounced in the Low group and not present in Control. The soaking protocol and ribose concentrations were chosen based on previous literature showing increases in AGEs. Therefore, we are confident changes noted here are due to the presence of AGEs and the resulting non-enzymatic crosslinks. Because soaking was performed in appropriately buffered solutions, decreased MMR in the High group relative to Control are expected to occur due to glycation of collagen rather than changes in mineral content. We suspect that perturbations in collagen structure due to the presence of non-enzymatic crosslinks are causing the differences in the area of the Amide I band between groups. Given the changes in MMR with glycation, future studies investigating models where AGEs are likely present should be cautious in their interpretation of MMR if it is not supported by other measures of mineralization. The lack of significant differences between groups for RPI and fracture toughness parameters may be due to the small sample size (n=4-5 per group) and biological variations associated with mechanical techniques. However, the sample size was adequate to assess correlations between Raman and RPI due to the co-localized measurements in each sample (n=70). The positive correlation between CID and MMR was expected given AGEs have been shown to reduce creep behavior and since MMR is decreased by AGEs. However, the correlation is weak which is likely due to the overall small non-significant effect in CID compared to its variation. The correlation between MMR and initiation toughness similarly suggests that as AGEs reduce MMR, KIc decreases which is known to occur with glycation. While the correlation did not reach significance (p=0.063), the trend is compelling given the small sample size (n=11) and the use of Raman data from an adjacent beam from the same sample rather than the beam used to measure KIc. In conclusion, MMR changes in response to in vitro glycation and these changes are correlated to CID and possibly to KIc. Deconvolution of the Amide I region into sub-peaks to determine which peak(s) are altered in the presence of AGEs is an important next step to developing a spectroscopic technique that can assess the presence of AGEs and is recommended in future work. Significance: Correlations were performed between Raman spectroscopy, Reference Point Indentation, and fracture toughness measurements to evaluate the ability of perturbations in the Amide I band to explain glycation-induced changes in tissue mechanics. Non-enzymatic glycation is an important determinant of bone quality especially in aging and diabetic patients and understanding the specific roles composition and microscale mechanics play in determining how non-enzymatic glycation affects fracture toughness may lead to new therapeutic targets.