A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.

Acidosis induces significant changes to the murine supraspinatus enthesis organic matrix.

Rotator cuff pathology is a common musculoskeletal condition that disproportionately affects older adults, as well as patients with diabetes mellitus and chronic kidney disease. It is known that increased age and kidney dysfunction have been correlated to acidotic states, which may be related to the increased incidence of rotator cuff injury. In order to investigate the potential relationship between acidosis and rotator cuff composition and mechanics, this study utilizes a 14-day murine model of metabolic acidosis and examines the effects on the supraspinatus tendon-humeral head attachment complex. The elastic matrix in the enthesis exhibited significant changes beginning at day 3 of acidosis exposure. At day 3 and day 7 timepoints, there was a decrease in collagen content seen in both mineralized and unmineralized tissue as well as a decrease in mineral:matrix ratio. There is also evidence of both mineral dissolution and reprecipitation as buffering ions continually promote pH homeostasis. Mechanical properties of the tendon-to-bone attachment were studied; however, no significant changes were elicited in this 14-day model of acidosis. These findings suggest that acidosis can result in significant changes in enthesis composition over the course of 14 days; however, enthesis mechanics may be more structurally mediated rather than affected by compositional changes.

PRG4 deficiency in mice alters skeletal structure, mechanics, and calvarial osteoclastogenesis, and rhPRG4 inhibits in vitro osteoclastogenesis.

Osteoporosis is a chronic disease characterized by reduced bone mass and increased fracture risk, estimated to affect over 10 million people in the United States alone. Drugs used to treat bone loss often come with significant limitations and/or long-term safety concerns. Proteoglycan-4 (PRG4, also known as lubricin) is a mucin-like glycoprotein best known for its boundary lubricating function of articular cartilage. In more recent years, it has been shown that PRG4 has anti-inflammatory properties, contributes to the maintenance of subchondral bone integrity, and patients with PRG4 mutations are osteopenic. However, it remains unknown how PRG4 impacts mechanical and material properties of bone. Therefore, our objective was to perform a phenotyping study of bone in a Prg4 gene trap (GT) mouse (PRG4 deficient). We found that femurs of Prg4 GT mice have altered mechanical, structural, and material properties relative to wildtype littermates. Additionally, Prg4 GT mice have a greater number of calvarial osteoclasts than wildtype mice, but do not have a notable inflammatory serum profile. Finally, Prg4 GT mice do not have an altered rate of bone formation, and exogenous recombinant human PRG4 (rhPRG4) administration inhibited osteoclastogenesis in vitro, suggesting that the skeletal phenotype may be due to changes in bone resorption. Overall, this work demonstrates that PRG4 deficiency affects several integral properties of bone structure, mechanics, and skeletal cell activity, and provides the foundation and insight toward future work evaluating PRG4 as a potential therapeutic target in treating bone loss.

Characterisation of the forest cobra (Naja melanoleuca) venom using a multifaceted mass spectrometric-based approach.

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated 'omics' and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

Effect of stress on the dissolution/crystallization of apatite in aqueous solution: a thermochemical equilibrium study.

Bone mineralization is critical to maintaining tissue mechanical function. The application of mechanical stress via exercise promotes bone mineralization via cellular mechanotransduction and increased fluid transport through the collagen matrix. However, due to its complex composition and ability to exchange ions with the surrounding body fluids, bone mineral composition and crystallization is also expected to respond to stress. Here, a combination of data from materials simulations, namely density functional theory and molecular dynamics, and experimental studies were input into an equilibrium thermodynamic model of bone apatite under stress in an aqueous solution based on the theory of thermochemical equilibrium of stressed solids. The model indicated that increasing uniaxial stress induced mineral crystallization. This was accompanied by a decrease in calcium and carbonate integration into the apatite solid. These results suggest that weight-bearing exercises can increase tissue mineralization via interactions between bone mineral and body fluid independent of cell and matrix behaviours, thus providing another mechanism by which exercise can improve bone health. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Metabolic Acidosis Results in Sexually Dimorphic Response in the Heart Tissue.

Metabolic acidosis (MA) is a highly prevalent disorder in a significant proportion of the population, resulting from imbalance in blood pH homeostasis. The heart, being an organ with very low regenerative capacity and high metabolic activity, is vulnerable to chronic, although low-grade, MA. To systematically characterize the effect of low-grade MA on the heart, we treated male and female mice with NH4Cl supplementation for 2 weeks and analyzed their blood chemistry and transcriptomic signature of the heart tissue. The reduction of pH and plasma bicarbonate levels without an associated change in anion gap indicated a physiological manifestation of low-grade MA with minimal respiratory compensation. On transcriptomic analysis, we observed changes in cardiac-specific genes with significant gender-based differences due to MA. We found many genes contributing to dilated cardiomyopathy to be altered in males, more than in females, while cardiac contractility and Na/K/ATPase-Src signaling were affected in the opposite way. Our model presents a systems-level understanding of how the cardiovascular tissue is affected by MA. As low-grade MA is a common ailment with many dietary and pharmaceutical interventions, our work presents avenues to limit chronic cardiac damage and disease manifestation, as well as highlighting the sex differences in MA-induced cardiovascular damage.

Efficient O-demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes.

A crucial reaction in harnessing renewable carbon from lignin is O-demethylation. We demonstrate the selective O-demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean O-demethylation of lignin-derived aromatics.

Cardiac ultrastructure inspired matrix induces advanced metabolic and functional maturation of differentiated human cardiomyocytes.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

The Magnet Is Sometimes "Off"-Practical Strategies for Optimizing Challenging Musculoskeletal MR Imaging.

To describe practical solutions to the unique technical challenges of musculoskeletal magnetic resonance imaging, including off-isocenter imaging, artifacts from motion and metal prostheses, small field-of-view imaging, and non-conventional scan angles and slice positioning. Unique challenges of musculoskeletal magnetic resonance imaging require a collaborative approach involving radiologists, physicists, and technologists utilizing optimized magnetic resonance protocols, specialized coils, and unique patient positioning, in order to reliably diagnose critical musculoskeletal MR image findings.

The Effects of Serum Removal on Gene Expression and Morphological Plasticity Markers in Differentiated SH-SY5Y Cells.

Despite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of ßIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.

Beyond dissolution: Xerostomia rinses affect composition and structure of biomimetic dental mineral in vitro.

Xerostomia, known as dry mouth, is caused by decreased salivary flow. Treatment with lubricating oral rinses provides temporary relief of dry mouth discomfort; however, it remains unclear how their composition affects mineralized dental tissues. Therefore, the objective of this study was to analyze the effects of common components in xerostomia oral rinses on biomimetic apatite with varying carbonate contents. Carbonated apatite was synthesized and exposed to one of the following solutions for 72 hours at varying pHs: water-based, phosphorus-containing (PBS), mucin-like containing (MLC), or fluoride-containing (FC) solutions. Post-exposure results indicated that apatite mass decreased irrespective of pH and solution composition, while solution buffering was pH dependent. Raman and X-ray diffraction analysis showed that the addition of phosphorus, mucin-like molecules, and fluoride in solution decreases mineral carbonate levels and changed the lattice spacing and crystallinity of bioapatite, indicative of dissolution/recrystallization processes. The mineral recrystallized into a less-carbonated apatite in the PBS and MLC solutions, and into fluorapatite in FC. Tap water did not affect the apatite lattice structure suggesting formation of a labile carbonate surface layer on apatite. These results reveal that solution composition can have varied and complex effects on dental mineral beyond dissolution, which can have long term consequences on mineral solubility and mechanics. Therefore, clinicians should consider these factors when advising treatments for xerostomia patients.

X-ray diffraction and in situ pressurization of dentine apatite reveals nanocrystal modulus stiffening upon carbonate removal.

Bone-like materials comprise carbonated-hydroxyapatite nanocrystals (c-Ap) embedding a fibrillar collagen matrix. The mineral particles stiffen the nanocomposite by tight attachment to the protein fibrils creating a high strength and toughness material. The nanometer dimensions of c-Ap crystals make it very challenging to measure their mechanical properties. Mineral in bony tissues such as dentine contains 2~6 wt.% carbonate with possibly different elastic properties as compared with crystalline hydroxyapatite. Here we determine strain in biogenic apatite nanocrystals by directly measuring atomic deformation in pig dentine before and after removing carbonate. Transmission electron microscopy revealed the platy 3D morphology while atom probe tomography revealed carbon inside the calcium rich domains. High-energy X-ray diffraction in combination with in situ hydrostatic pressurization quantified reversible c-Ap deformations. Crystal strains differed between annealed and ashed (decarbonated) samples, following 1 or 10 h heating at 250 °C or 550 °C respectively. Measured bulk moduli (K) and a-/c-lattice deformation ratios (η) were used to generate synthetic Ksyn and ηsyn identifying the most likely elastic constants C33 and C13 for c-Ap. These were then used to calculate the nanoparticle elastic moduli. For ashed samples, we find an average E11=107 GPa and E33 =128 GPa corresponding to ~5% and ~17% stiffening of the a-/c-axes of the nanocrystals as compared with the biogenic nanocrystals in annealed samples. Ashed samples exhibit ~10% lower Poisson's ratios as compared with the 0.25~0.36 range of carbonated apatite. Carbonate in c-Ap may therefore serve for tuning local deformability within bony tissues. STATEMENT OF SIGNIFICANCE: Carbonated apatite nanoparticles, typical for bony tissues, stiffen the network of collagen fibrils. However, it is not known if the biogenic apatite mechanical (elastic) properties differ from those of geologic mineral counterparts. Indeed the tiny dimensions and variable carbonate composition may have strong effects on deformation resistance. The present study provides experimental measurements of the elastic constants which we use to estimate Young's moduli and Poisson's ratio values. Comparison between ashed and annealed dentine samples quantifies the properties of both carbonated and decarbonated apatite nanocrystals. The results reveal fundamental attributes of bony mineral and showcase the additive advantages of combining X-ray diffraction with in situ hydrostatic compression, backed by atom probe and transmission electron microscopy tomography.

Inhibition of miR-29-3p isoforms via tough decoy suppresses osteoblast function in homeostasis but promotes intermittent parathyroid hormone-induced bone anabolism.

miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/ßcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.

Transcranial Magnetic Stimulation-Induced Plasticity Mechanisms: TMS-Related Gene Expression and Morphology Changes in a Human Neuron-Like Cell Model.

Transcranial Magnetic Stimulation (TMS) is a form of non-invasive brain stimulation, used to alter cortical excitability both in research and clinical applications. The intermittent and continuous Theta Burst Stimulation (iTBS and cTBS) protocols have been shown to induce opposite after-effects on human cortex excitability. Animal studies have implicated synaptic plasticity mechanisms long-term potentiation (LTP, for iTBS) and depression (LTD, for cTBS). However, the neural basis of TMS effects has not yet been studied in human neuronal cells, in particular at the level of gene expression and synaptogenesis. To investigate responses to TBS in living human neurons, we differentiated human SH-SY5Y cells toward a mature neural phenotype, and stimulated them with iTBS, cTBS, or sham (placebo) TBS. Changes in (a) mRNA expression of a set of target genes (previously associated with synaptic plasticity), and (b) morphological parameters of neurite outgrowth following TBS were quantified. We found no general effects of stimulation condition or time on gene expression, though we did observe a significantly enhanced expression of plasticity genes NTRK2 and MAPK9 24 h after iTBS as compared to sham TBS. This specific effect provides unique support for the widely assumed plasticity mechanisms underlying iTBS effects on human cortex excitability. In addition to this protocol-specific increase in plasticity gene expression 24 h after iTBS stimulation, we establish the feasibility of stimulating living human neuron with TBS, and the importance of moving to more complex human in vitro models to understand the underlying plasticity mechanisms of TBS stimulation.

The Association Between Endotracheal Tube Size and Aspiration (During Flexible Endoscopic Evaluation of Swallowing) in Acute Respiratory Failure Survivors.

OBJECTIVES: To determine whether a modifiable risk factor, endotracheal tube size, is associated with the diagnosis of postextubation aspiration in survivors of acute respiratory failure. DESIGN: Prospective cohort study. SETTING: ICUs at four academic tertiary care medical centers. PATIENTS: Two hundred ten patients who were at least 18 years old, admitted to an ICU, and mechanically ventilated with an endotracheal tube for longer than 48 hours were enrolled. INTERVENTIONS: Within 72 hours of extubation, all patients received a flexible endoscopic evaluation of swallowing examination that entailed administration of ice, thin liquid, thick liquid, puree, and cracker boluses. Patient demographics, treatment variables, and hospital outcomes were abstracted from the patient's medical records. Endotracheal tube size was independently selected by the patient's treating physicians. MEASUREMENTS AND MAIN RESULTS: For each flexible endoscopic evaluation of swallowing examination, laryngeal pathology was evaluated, and for each bolus, a Penetration Aspiration Scale score was assigned. Aspiration (Penetration Aspiration Scale score ≥ 6) was further categorized into nonsilent aspiration (Penetration Aspiration Scale score = 6 or 7) and silent aspiration (Penetration Aspiration Scale score = 8). One third of patients (n = 68) aspirated (Penetration Aspiration Scale score ≥ 6) on at least one bolus, 13.6% (n = 29) exhibited silent aspiration, and 23.8% (n = 50) exhibited nonsilent aspiration. In a multivariable analysis, endotracheal tube size (≤ 7.5 vs ≥ 8.0) was significantly associated with patients exhibiting any aspiration (Penetration Aspiration Scale score ≥ 6) (p = 0.016; odds ratio = 2.17; 95% CI 1.14-4.13) and with risk of developing laryngeal granulation tissue (p = 0.02). CONCLUSIONS: Larger endotracheal tube size was associated with increased risk of aspiration and laryngeal granulation tissue. Using smaller endotracheal tubes may reduce the risk of postextubation aspiration.

Iatrogenic humeral anatomic neck fracture after intraosseous vascular access.

Intraosseous infusion has become a key tool in the resuscitation of critically ill or injured patients, both in pre-hospital settings and in emergency departments. Intraosseous access is obtained through the percutaneous placement of a needle into the medullary space of a bone, thereby allowing access into the systemic venous circulation via the medullary space, which is essential to treat patients in shock, cardiac arrest, airway compromise, or major trauma. This becomes critically important when obtaining conventional intravenous access is difficult or impossible. Few cases of iatrogenic fracture have been reported for intraosseous access in the tibia and no case to-date has been reported of iatrogenic fracture secondary to humeral access. We report a case of a 55-year-old patient being resuscitated emergently with proximal humeral intraosseous infusion for cardiac and respiratory arrest secondary to status epilepticus. After successful resuscitation and removal of the intraosseous cannula, the patient noted new-onset shoulder pain. The patient was ultimately diagnosed with an iatrogenic fracture of the anatomic neck of the humerus through the intraosseous needle tract when the appropriate history was obtained in conjunction with cross-sectional imaging. As the use of intraosseous access expands, such fractures may well be seen more frequently. Intraosseous access is limited to the period of resuscitation and the cannula is often not present at the time of imaging. It is important for radiologists to recognize the findings related to intraosseous access as well as this complication with its characteristic locations and morphology.

Multiscale effects of spaceflight on murine tendon and bone.

Despite a wealth of data on the effects of spaceflight on tendons and bones, little is known about its effects on the interfacial tissue between these two structures, the enthesis. Mice were sent to space on three separate missions: STS-131, STS-135, and Bion-M1 to determine how spaceflight affects the composition, structure, mechanics, and gene expression of the humerus-supraspinatus and calcaneus-Achilles entheses. At the nanoscale, spaceflight resulted in decreased carbonate levels in the bone, likely due to increased remodeling, as suggested by increased expression of genes related to osteoclastogenesis (CatK, Tnfsf11) and mature osteoblasts (Col1, Osc). Tendons showed a shift in collagen fibril size towards smaller diameters that may have resulted from increased expression of genes related to collagen degradation (Mmp3, Mmp13). These nanoscale changes did not result in micro- and milliscale changes to the structure and mechanics of the enthesis. There were no changes in bone volume, trabecular structure, failure load, or stiffness with spaceflight. This lack of tissue-level change may be anatomy based, as extremities may be less sensitive to spaceflight than central locations such as vertebrae, yet results highlight that the tendon enthesis may be robust against negative effects of spaceflight.

Sister Mary Joseph Nodule in Advanced Pancreatic Adenocarcinoma Identified on 18F-FDG PET/MRI.

Sister Mary Joseph nodule is an umbilical metastatic lesion typically originating from gastrointestinal or gynecologic malignancies. In pancreatic cancer, Sister Mary Joseph nodule is a sign of advanced disease with an associated poor prognosis. 18F-FDG PET/MRI provides an imaging tool in the identification of Sister Mary Joseph nodule and helps to improve the staging of pancreatic cancer.