Browsing by Author "Berman, Alycia G."
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Item 6'-Methoxy Raloxifene-analog enhances mouse bone properties with reduced estrogen receptor binding(Elsevier, 2020-01-17) Powell, Katherine M.; Brown, Alexa P.; Skaggs, Cayla G.; Pulliam, Alexis N.; Berman, Alycia G.; Deosthale, Padmini; Plotkin, Lilian I.; Allen, Matthew R.; Williams, David R.; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyRaloxifene (RAL) is an FDA-approved drug used to treat osteoporosis in postmenopausal women. RAL suppresses bone loss primarily through its role as a selective estrogen receptor modulator (SERM). This hormonal estrogen therapy promotes unintended side effects, such as hot flashes and increased thrombosis risk, and prevents the drug from being used in some patient populations at-risk for fracture, including children with bone disorders. It has recently been demonstrated that RAL can have significant positive effects on overall bone mechanical properties by binding to collagen and increasing bone tissue hydration in a cell-independent manner. A Raloxifene-Analog (RAL-A) was synthesized by replacing the 6-hydroxyl substituent with 6-methoxy in effort to reduce the compound's binding affinity for estrogen receptors (ER) while maintaining its collagen-binding ability. It was hypothesized that RAL-A would improve the mechanical integrity of bone in a manner similar to RAL, but with reduced estrogen receptor binding. Molecular assessment showed that while RAL-A did reduce ER binding, downstream ER signaling was not completely abolished. In-vitro, RAL-A performed similarly to RAL and had an identical concentration threshold on osteocyte cell proliferation, differentiation, and function. To assess treatment effect in-vivo, wildtype (WT) and heterozygous (OIM+/-) female mice from the Osteogenesis Imperfecta (OI) murine model were treated with either RAL or RAL-A from 8 weeks to 16 weeks of age. There was an untreated control group for each genotype as well. Bone microarchitecture was assessed using microCT, and mechanical behavior was assessed using 3-point bending. Results indicate that both compounds produced analogous gains in tibial trabecular and cortical microarchitecture. While WT mechanical properties were not drastically altered with either treatment, OIM+/- mechanical properties were significantly enhanced, most notably, in post-yield properties including bone toughness. This proof-of-concept study shows promising results and warrants the exploration of additional analog iterations to further reduce ER binding and improve fracture resistance.Item Bone Quality and Quantity are Mediated by Mechanical Stimuli(Springer, 2016-09) Berman, Alycia G.; Wallace, Joseph M.; Department of Biomedical Engineering, School of EngineeringPrevention of fracture through improved bone mechanical strength is of great importance given the large number of bone disease-related fractures each year, the decreased quality of life associated with fractures, and the large anticipated increase in fracture incidence over the upcoming years due to the aging population. Exercise and other forms of mechanical stimulation have been shown to increase bone mass, suggesting improved strength. However, while bone mass is a good indicator of strength, other components (such as bone quality) also contribute to bone mechanical integrity. While increased bone mass has been explored considerably using both exercise and targeted loading models, the role of mechanical stimulation in altering bone quality has been explored to a lesser degree. Understanding how to improve both the quantity and quality of bone is critical to increasing fracture resistance. Herein, we discuss quantity and quality-based improvements that have been observed using both exercise and targeted loading models of bone adaptation.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 Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice(Taylor & Francis, 2022) Berman, Alycia G.; Damrath, John G.; Hatch, Jennifer; Pulliam, Alexis N.; Powell, Katherine M.; Hinton, Madicyn; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyRaloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements. To do so, structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age. Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present. In conclusion, RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.Item Influence of Mechanical Stimulation on the Quantity and Quality of Bone During Modeling(2016) Berman, Alycia G.; Wallace, Joseph; Na, Sungsoo; Li, Jiliang; Yoshida, KenSkeletal fractures due to bone disease impact an estimated 1.5 million Americans per year, creating a large economic burden on our society. Treatment of bone diseases prior to fracture often involves bisphosphonates (current gold-standard in osteoporosis care and prevention). Although bisphosphonates decrease fracture incidence, they often improve bone mass without regard for bone quality. Thus, although bisphosphonates increase the amount of bone present, the inherent bone material strength often decreases, creating a trade-off that increases the risk of atypical fractures after long-term use. This trade-off demonstrates the need for a treatment that targets both bone quality AND quantity. Although bone quality is important, the components of bone that contribute to bone quality are incompletely understood, making it difficult to create new pharmacological agents. With this in mind, my particular area of interest is in understanding how mechanical stimuli protects the formation of bone, leading to improved bone quality. Initially, this area was explored through use of tibial loading in a disease mouse model (osteolathyrism, induced by injection of beta-aminoproprionitrile) as a means of assessing how the body is able to compensate for decreased bone quality. The results of the BAPN and tibial loading studies indicated that injecting mice with BAPN may not be the ideal method to induce osteolathyrism. However, other intriguing results from the BAPN studies then led us into an exploration of how tibial loading itself contributes to bone quality.Item A Ligand Trap of the Activin Receptor Type IIA Inhibits Osteoclast Stimulation of Bone Remodeling in Diabetic Mice with Chronic Kidney Disease(Elsevier, 2017-01) Sugatini, Toshifumi; Agapova, Olga A.; Fang, Yifu; Berman, Alycia G.; Wallace, Joseph M.; Malluche, Hartmut H.; Faugere, Marie-Claude; Smith, William; Sung, Victoria; Hruska, Keith A.; Department of Biomedical Engineering, School of Engineering and TechnologyDysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat–fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat–fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A–enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin–A induced osteoclastogenesis.Item Muscle contraction induces osteogenic levels of cortical bone strain despite muscle weakness in a mouse model of Osteogenesis Imperfecta(Elsevier, 2020) Berman, Alycia G.; Organ, Jason M.; Allen, Matthew R.; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyMechanical interactions between muscle and bone have long been recognized as integral to bone integrity. However, few studies have directly measured these interactions within the context of musculoskeletal disease. In this study, the osteogenesis imperfecta murine model (oim/oim) was utilized because it has both reduced bone and muscle properties, allowing direct assessment of whether weakened muscle is able to engender strain on weakened bone. To do so, a strain gauge was attached to the tibia of healthy and oim/oim mice, muscles within the posterior quadrant of the lower hind limb were stimulated, and bone strain during muscle contraction was measured. Results indicated that the relationship between maximum muscle torque and maximum engendered strain is altered in oim/oim bone, with less torque required to engender strain compare to wild-type and heterozygous mice. Maximum muscle torque at 150 Hz stimulation frequency was able to engender ~1500 μɛ in oim/oim animals. However, even though the strain engendered in the oim/oim mice was high relative to historical bone formation thresholds, the maximum strain values were still significantly lower than that of the wild-type mice. These results are promising in that they suggest that muscle stimulation may be a viable means of inducing bone formation in oim/oim and potentially other disease models where muscle weakness/atrophy exist.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.Item Raloxifene reduces skeletal fractures in an animal model of osteogenesis imperfecta(Elsevier, 2016) Berman, Alycia G.; Wallace, Joseph M.; Bart, Zachary R.; Allen, Matthew R.; Anatomy and Cell Biology, School of MedicineOsteogenesis imperfecta (OI) is a genetic disease of Type I collagen and collagen-associated pathways that results in brittle bone behavior characterized by fracture and reduced mechanical properties. Based on previous work in our laboratory showing that raloxifene (RAL) can significantly improve bone mechanical properties through non-cellular mechanisms, we hypothesized that raloxifene would improve the mechanical properties of OI bone. In experiment 1, tibiae from female wild type (WT) and homozygous oim mice were subjected to in vitro soaking in RAL followed by mechanical tests. RAL soaking resulted in significantly higher post-yield displacement (+75% in WT, +472% in oim; p<0.004), with no effect on ultimate load or stiffness, in both WT and oim animals. In experiment 2, eight-week old WT and oim male mice were treated for eight weeks with saline vehicle (VEH) or RAL. Endpoint measures included assessment of in vivo skeletal fractures, bone density/geometry and mechanical properties. In vivo skeletal fractures of the femora, assessed by micro CT imaging, were significantly lower in oim-RAL (20%) compared to oim-VEH (48%, p=0.047). RAL led to significantly higher DXA-based BMD (p<0.01) and CT-based trabecular BV/TV in both WT and oim animals compared to those treated with VEH. Fracture toughness of the femora was lower in oim mice compared to WT and improved with RAL in both genotypes. These results suggest that raloxifene reduces the incidence of fracture in this mouse model of oim. Furthermore, they suggest that raloxifene's effects may be the result of both cellular (increased bone mass) and non-cellular (presumably changes in hydration) mechanisms, raising the possibility of using raloxifene, or related compounds, as a new approach for treating bone fragility associated with OI.