Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function.

Dysregulation 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.

Cardiac hypertrophy is closely linked to impaired fatty acid oxidation, but the molecular basis of this link is unclear. Here, we investigated the loss of an obligate enzyme in mitochondrial long-chain fatty acid oxidation, carnitine palmitoyltransferase 2 (CPT2), on muscle and heart structure, function, and molecular signatures in a muscle- and heart-specific CPT2-deficient mouse (Cpt2) model. CPT2 loss in heart and muscle reduced complete oxidation of long-chain fatty acids by 87 and 69%, respectively, without altering body weight, energy expenditure, respiratory quotient, or adiposity. Cpt2M mice developed cardiac hypertrophy and systolic dysfunction, evidenced by a 5-fold greater heart mass, 60-90% reduction in blood ejection fraction relative to control mice, and eventual lethality in the absence of cardiac fibrosis. The hypertrophy-inducing mammalian target of rapamycin complex 1 (mTORC1) pathway was activated in Cpt2M hearts; however, daily rapamycin exposure failed to attenuate hypertrophy in Cpt2M mice. Lysine acetylation was reduced by ∼50% in Cpt2M hearts, but trichostatin A, a histone deacetylase inhibitor that improves cardiac remodeling, failed to attenuate Cpt2M hypertrophy. Strikingly, a ketogenic diet increased lysine acetylation in Cpt2M hearts 2.3-fold compared with littermate control mice fed a ketogenic diet, yet it did not improve cardiac hypertrophy. Together, these results suggest that a shift away from mitochondrial fatty acid oxidation initiates deleterious hypertrophic cardiac remodeling independent of fibrosis. The data also indicate that CPT2-deficient hearts are impervious to hypertrophy attenuators, that mitochondrial metabolism regulates cardiac acetylation, and that signals derived from alterations in mitochondrial metabolism are the key mediators of cardiac hypertrophic growth.

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Scope 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 have 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. Conclusions 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.

An abdominal aortic aneurysm (AAA), defined as a pathological expansion of the largest artery in the abdomen, is a common vascular disease that frequently leads to death if rupture occurs. Once diagnosed, clinicians typically evaluate the rupture risk based on maximum diameter of the aneurysm, a limited metric that is not accurate for all patients. In this study, we worked to evaluate additional distinguishing factors between growing and stable murine aneurysms toward the aim of eventually improving clinical rupture risk assessment. With the use of a relatively new mouse model that combines surgical application of topical elastase to cause initial aortic expansion and a lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), in the drinking water, we were able to create large AAAs that expanded over 28 days. We further sought to develop and demonstrate applications of advanced imaging approaches, including four-dimensional ultrasound (4DUS), to evaluate alternative geometric and biomechanical parameters between 1) growing AAAs, 2) stable AAAs, and 3) nonaneurysmal control mice. Our study confirmed the reproducibility of this murine model and found reduced circumferential strain values, greater tortuosity, and increased elastin degradation in mice with aneurysms. We also found that expanding murine AAAs had increased peak wall stress and surface area per length compared with stable aneurysms. The results from this work provide clear growth patterns associated with BAPN-elastase murine aneurysms and demonstrate the capabilities of high-frequency ultrasound. These data could help lay the groundwork for improving insight into clinical prediction of AAA expansion. NEW & NOTEWORTHY This work characterizes a relatively new murine model of abdominal aortic aneurysms (AAAs) by quantifying vascular strain, stress, and geometry. Furthermore, Green-Lagrange strain was calculated with a novel mapping approach using four-dimensional ultrasound. We also compared growing and stable AAAs, finding peak wall stress and surface area per length to be most indicative of growth. In all AAAs, strain and elastin health declined, whereas tortuosity increased.

With approximately 48,000 attributed deaths in 2017, the opioid overdose is now the leading cause of death amongst Americans under the age of 50. The overdose process can be interrupted by the administration of naloxone, a safe and effective opiate antagonist that can reverse the effects of overdose and minimizing the delay in administering the antidote is critical in preventing permanent damage to patients. A closed-loop implantable drug delivery system is an ideal solution to minimize the response time, however, they often feature complex designs that are expensive to fabricate and require a more invasive surgical implantation. Here we propose a simple, low-cost, minimally-invasive automatic antidote delivery device (A2D2) that can administer a large dose of naloxone upon detection of overdose-induced respiratory failure. The subcutaneously placed device can be activated using an externally applied time varying magnetic field from a wearable device. Using a custom magnetic field generator, we were able to release the drug within 10 s. Our bench-top evaluation showed that A2D2 can release 1.9 mg of powdered drug within 60 s and up to 8.8 mg in 600 s. We also performed in vivo evaluation to demonstrate rapid drug releasing capability in the subcutaneous space of mice. However, we saw a small amount of leakage (1.75% of payload) over the course of 1000 h of simulated implantation. Thus, additional research is needed to verify the long term stability of our device and to demonstrate the closed-loop release mechanism to revive overdosed animals. Nevertheless, our preliminary results show the potential of using a simple, low-cost, subcutaneous device for emergency drug delivery application.

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