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.

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.

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.

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.

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.

The multiscale structural and mechanical effects of mouse supraspinatus muscle unloading on the mature enthesis.

The musculoskeletal system is sensitive to its loading environment; this is of particular concern under conditions such as disuse, paralysis, and extended-duration space flight. Although structural and mechanical changes to tendon and bone following paralysis and disuse are well understood, there is a pressing need to understand how this unloading affects the bone-tendon interface (enthesis); the location most prone to tears and injury. We therefore elucidated these effects of unloading in the entheses of adult mice shoulders that were paralyzed for 21 days by treatment with botulinum toxin A. Unloading significantly increased the extent of mechanical failure and was associated with structural changes across hierarchical scales. At the millimeter scale, unloading caused bone loss. At the micrometer scale, unloading decreased bioapatite crystal size and crystallographic alignment in the enthesis. At the nanometer scale, unloading induced compositional changes that stiffened the bioapatite/collagen composite tissue. Mathematical modeling and mechanical testing indicated that these factors combined to increase local elevations of stress while decreasing the ability of the tissue to absorb energy prior to failure, thereby increasing injury risk. These first observations of the multiscale effects of unloading on the adult enthesis provide new insight into the hierarchical features of structure and composition that endow the enthesis with increased resistance to failure. STATEMENT OF SIGNIFICANCE: The musculoskeletal system is sensitive to its loading environment; this is of particular concern under conditions such as disuse, paralysis, and extended-duration space flight. Although changes to tendon and bone following paralysis are understood, there is a pressing need to clarify how unloading affects the bone-tendon interface (enthesis), which is the location most prone to tears and injury. We elucidated the effects of enthesis unloading in adult mice shoulders showing, for the first time, that unloading significantly increased the risk and extent of mechanical failure and was associated with structural changes across hierarchical scales. These observations provide new insight into the hierarchical features of structure and composition that endow the enthesis with resilience. This knowledge can be used to develop more targeted treatments to improve mobility and function.

Effects of spaceflight on the muscles of the murine shoulder.

Mechanical loading is necessary for the development and maintenance of the musculoskeletal system. Removal of loading via microgravity, paralysis, or bed rest leads to rapid loss of muscle mass and function; however, the molecular mechanisms that lead to these changes are largely unknown, particularly for the spaceflight (SF) microgravity environment. Furthermore, few studies have explored these effects on the shoulder, a dynamically stabilized joint with a large range of motion; therefore, we examined the effects of microgravity on mouse shoulder muscles for the 15-d Space Transportation System (STS)-131, 13-d STS-135, and 30-d Bion-M1 missions. Mice from STS missions were euthanized within 4 h after landing, whereas mice from the Bion-M1 mission were euthanized within 14 h after landing. The motion-generating deltoid muscle was more sensitive to microgravity than the joint-stabilizing rotator cuff muscles. Mice from the STS-131 mission exhibited reduced myogenic (Myf5 and -6) and adipogenic (Pparg, Cebpa, and Lep) gene expression, whereas either no change or an increased expression of these genes was observed in mice from the Bion-M1 mission. In summary, muscle responses to microgravity were muscle-type specific, short-duration SF caused dramatic molecular changes to shoulder muscles and responses to reloading upon landing were rapid.-Shen, H., Lim, C., Schwartz, A. G., Andreev-Andrievskiy, A., Deymier, A. C., Thomopoulos, S. Effects of spaceflight on the muscles of the murine shoulder.

Protein-free formation of bone-like apatite: New insights into the key role of carbonation.

The nanometer-sized plate-like morphology of bone mineral is necessary for proper bone mechanics and physiology. However, mechanisms regulating the morphology of these mineral nanocrystals remain unclear. The dominant hypothesis attributes the size and shape regulation to organic-mineral interactions. Here, we present data supporting the hypothesis that physicochemical effects of carbonate integration within the apatite lattice control the morphology, size, and mechanics of bioapatite mineral crystals. Carbonated apatites synthesized in the absence of organic molecules presented plate-like morphologies and nanoscale crystallite dimensions. Experimentally-determined crystallite size, lattice spacing, solubility and atomic order were modified by carbonate concentration. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations predicted changes in surface energy and elastic moduli with carbonate concentration. Combining these results with a scaling law predicted the experimentally observed scaling of size and energetics with carbonate concentration. The experiments and models describe a clear mechanism by which crystal dimensions are controlled by carbonate substitution. Furthermore, the results demonstrate that carbonate substitution is sufficient to drive the formation of bone-like crystallites. This new understanding points to pathways for biomimetic synthesis of novel, nanostructured biomaterials.

Micro-mechanical properties of the tendon-to-bone attachment.

The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue that connects stiff bone to compliant tendon. The attachment site at the micrometer scale exhibits gradients in mineral content and collagen orientation, which likely act to minimize stress concentrations. The physiological micromechanics of the attachment thus define resultant performance, but difficulties in sample preparation and mechanical testing at this scale have restricted understanding of structure-mechanical function. Here, microscale beams from entheses of wild type mice and mice with mineral defects were prepared using cryo-focused ion beam milling and pulled to failure using a modified atomic force microscopy system. Micromechanical behavior of tendon-to-bone structures, including elastic modulus, strength, resilience, and toughness, were obtained. Results demonstrated considerably higher mechanical performance at the micrometer length scale compared to the millimeter tissue length scale, describing enthesis material properties without the influence of higher order structural effects such as defects. Micromechanical investigation revealed a decrease in strength in entheses with mineral defects. To further examine structure-mechanical function relationships, local deformation behavior along the tendon-to-bone attachment was determined using local image correlation. A high compliance zone near the mineralized gradient of the attachment was clearly identified and highlighted the lack of correlation between mineral distribution and strain on the low-mineral end of the attachment. This compliant region is proposed to act as an energy absorbing component, limiting catastrophic failure within the tendon-to-bone attachment through higher local deformation. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue. STATEMENT OF SIGNIFICANCE: The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue with features at a numerous scales that dissipate stress concentrations between compliant tendon and stiff bone. At the micrometer scale, the enthesis exhibits gradients in collagen and mineral composition and organization. However, the physiological mechanics of the enthesis at this scale remained unknown due to difficulty in preparing and testing micrometer scale samples. This study is the first to measure the tensile mechanical properties of the enthesis at the micrometer scale. Results demonstrated considerably enhanced mechanical performance at the micrometer length scale compared to the millimeter tissue length scale and identified a high-compliance zone near the mineralized gradient of the attachment. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue.

The concentration of stress at the rotator cuff tendon-to-bone attachment site is conserved across species.

The tendon-to-bone attachment site integrates two distinct tissues via a gradual transition in composition, mechanical properties, and structure. Outcomes of surgical repair are poor, in part because surgical repair does not recreate the natural attachment, and in part because the mechanical features that are most critical to mechanical and physiological functions have not been identified. We employed allometric analysis to resolve a paradox about how the architecture of the rotator cuff contributes to load transfer: whereas published data suggest that the mean muscle stresses expected at the tendon-to-bone attachment are conserved across species, data also show that the relative dimensions of key anatomical features vary dramatically, suggesting that the amplification of stresses at the interface between tendon and bone should also vary widely. However, a mechanical model that enabled a sensitivity analysis revealed that the degree of stress concentration was in fact highly conserved across species: the factors that most affected stress amplification were most highly conserved across species, while those that had a lower effect showed broad variation across a range of relative insensitivity. Results highlight how micromechanical factors can influence structure-function relationships and cross-species scaling over several orders of magnitude in animal size, and provide guidance on physiological features to emphasize in surgical and tissue engineered repair of the rotator cuff.

Tunability of collagen matrix mechanical properties via multiple modes of mineralization.

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering.