Tissue-level Properties Research Articles

Mechanical impact of regional structural deterioration and tissue-level compensation on proximal femur trabecular bone.

Osteoporosis-induced changes in bone structure and composition significantly reduce bone strength, particularly in the human proximal femur. This study examines how these changes affect the mechanical performance of trabecular bone to enhance diagnosis, prevention, and treatment strategies. A proximal femur sample was scanned using micro-CT at 40μm resolution. Five regions of interest were selected within the femoral head, femoral neck, and greater trochanter. Structural models simulating various stages of osteoporosis were created using image processing software. Micro-finite element analysis evaluated the mechanical properties of trabecular bone under different conditions of structural deterioration and tissue-level elastic modulus variations. The combined effects of structural deterioration and tissue-level mechanical properties on trabecular bone mechanical performance were further analyzed. The mechanical performance of trabecular bone generally follows a power-law relationship with its microstructural characteristics. However, in any specific region, the apparent mechanical properties linearly decrease with structural deterioration. The femoral neck and greater trochanter are more sensitive to structural deterioration than the femoral head. A 5% bone mass loss in the femoral head led to a 7% reduction in mechanical performance, while the femoral neck experienced a 12% loss. Increasing tissue-level elastic modulus improved mechanical performance, partially offsetting bone mass reduction effects. Trabecular bone in low bone mass regions is more affected by bone mass loss. Structural deterioration primarily reduces bone strength, but improvements in tissue-level properties can mitigate this effect, especially in early osteoporosis. Targeted assessments and interventions are crucial for effective management. Future research should explore heterogeneous deterioration models to better understand osteoporosis progression.

Differences in tissue-level properties as assessed by nano-scratching in patients with and without atypical femur fractures on long-term bisphosphonate therapy: a proof-of-concept pilot study.

Atypical femur fractures (AFFs) are a well-established complication of long-term bisphosphonate (BP) therapy, but their pathogenesis is not fully understood. Although many patients on long-term BP therapy have severe suppression of bone turnover (SSBT), not all such patients experience AFF, even though SSBT is a major contributor to AFF. Accordingly, we evaluated tissue level properties using nano-scratch testing of trans-iliac bone biopsy specimens in 12 women (6 with and 6 without AFF matched for age and race). Nano-scratch data were analyzed using a mixed-model ANOVA with volume-normalized scratch energy as a function of AFF (Yes or No), region (periosteal or endosteal), and a first-order interaction between region and AFF. Tukey post hoc analyses of the differences of least squared means of scratch energy were performed and reported as significant if p<.05. The volume-normalized scratch energy was 10.6% higher in AFF than in non-AFF patients (p=.003) and 17.9 % higher in the periosteal than in the endosteal region (p=.004). The differences in normalized scratch energy are suggestive of a higher hardness of the bone tissue after long-term BP therapy. The results of this study are consistent with other studies in the literature and demonstrate the efficacy of using Nano-Scratch technique to evaluate bone tissue that exhibits SSBT and AFF. Further studies using nano-scratch may help quantify and elucidate underlying mechanisms for the pathogenesis of AFF.

Biomechanical and biochemical changes in murine skin during development and aging

Aging leads to biochemical and biomechanical changes in skin, with biological and functional consequences. Despite extensive literature on skin aging, there is a lack of studies which investigate the maturation of the tissue and connect the microscopic changes in the skin to its macroscopic biomechanical behavior as it evolves over time. The present work addresses this knowledge gap using multiscale characterization of skin in a murine model considering newborn, adult and aged mice. Monotonic uniaxial loading, tension relaxation with change of bath, and loading to failure tests were performed on murine skin samples from different age groups, complemented by inflation experiments and atomic force microscopy indentation measurements. In parallel, skin samples were characterized using histological and biochemical techniques to assess tissue morphology, collagen organization, as well as collagen content and cross-linking. We show that 1-week-old skin differs across nearly all measured parameters from adult skin, showing reduced strain stiffening and tensile strength, a thinner dermis, lower collagen content and altered crosslinking patterns. Surprisingly, adult and aged skin were similar across most biomechanical parameters in the physiologic loading range, while aged skin had lower tensile strength and lower stiffening behavior at large force values. This correlates with altered collagen content and cross-links. Based on a computational model, differences in mechanocoupled stimuli in the skin of the different age groups were calculated, pointing to a potential biological significance of the age-induced biomechanical changes in regulating the local biophysical environment of dermal cells. Statement of significanceSkin microstructure and the emerging mechanical properties change with age, leading to biological, functional and health-related consequences. Despite extensive literature on skin aging, only very limited quantitative data are available on microstructural changes and the corresponding macroscopic biomechanical behavior as they evolve over time. This work provides a wide-range multiscale mechanical characterization of skin of newborn, adult and aged mice, and quantifies microstructural correlations in tissue morphology, collagen content, organization and cross-linking. Remarkably, aged skin retained normal hydration and normal biomechanical function in the physiological loading range but showed significantly reduced properties at super-physiological loading. Our data show that age-related microstructural differences have a profound effect not only on tissue-level properties but also on the cell-level biophysical environment.

Aging and injury affect nuclear shape heterogeneity in tendon.

Tissue level properties are commonly studied using histological stains assessed with qualitative scoring methods. As qualitative evaluation is typically insensitive, quantitative analysis provides additional information about pathological mechanisms, but cannot capture structural heterogeneity across cell subpopulations. However, molecular analyses of cell and nuclear behavior have identified that cell and more recently also nuclear shape are highly associated with cell function and malfunction. This study combined a Visually Aided Morpho-Phenotyping Image Recognition analysis that automatically segments cells based on their shape with an added capacity to further discriminate between cells in certain protein-rich extracellular matrix regions. We used tendon as a model system given the enormous changes in organization and cell and nuclear shape they undergo during aging and injury. Our results uncover that multiple shape modes of nuclei exist during maturity and aging in rat tendon and that distinct subgroups of cell nuclei shapes exist in proteoglycan-rich regions during aging. With injury, several immunomarkers (αSMA, CD31, CD146) were associated with more rounded shape modes. In human tendons, the cell nuclei at sites of injury were found to be more rounded relative to uninjured tissues. To conclude, the tendon tissue changes occurring during aging and injury could be associated with a variation in cell nuclear morphology and the appearance of various region-specific subpopulations. Thus, the methodologies developed allow for a deeper understanding of cell heterogeneity during tendon aging and injury and may be extended to study further clinical applications.

Association between nanoscale strains and tissue level nanoindentation properties in age-related hip-fractures

Measurement of the properties of bone as a material can happen in various length scales in its hierarchical and composite structure. The aim of this study was to test the tissue level properties of clinically-relevant human bone samples which were collected from donors belonging to three groups: ageing donors who suffered no fractures (Control); untreated fracture patients (Fx-Untreated) and patient who experienced hip fracture despite being treated with bisphosphonates (Fx-BisTreated). Tissue level properties were assessed by (a) nanoindentation and (b) synchrotron tensile tests (STT) where strains were measured at the ‘tissue’, ‘fibril’ and ‘mineral’ levels by using simultaneous Wide-angle - (WAXD) and Small angle- X-ray diffraction (SAXD). The composition was analysed by thermogravimetric analysis and material level endo- and exo-thermic reactions by differential scanning calorimetry (TGA/DSC3+). Irrespective of treatment fracture donors exhibited significantly lower tissue, fibril and mineral strain at the micro and nanoscale respectively and had a higher mineral content than controls. In nanoindentation only nanohardness was significantly greater for Controls and Fx-BisTreated versus Fx-Untreated. The other nanoindentation parameters did not vary significantly across the three groups. There was a highly significant positive correlation (p < 0.001) between organic content and tissue level strain behaviour. Overall hip-fractures were associated with lower STT nanostrains and it was behaviour measured by STT which proved to be a more effective approach for predicting fracture risk because evidently it was able to demonstrate the mechanical deficit for the bone tissue of the donors who had experienced fractures.

Establishment and Image based evaluation of a New Preclinical Rat Model of Osteoblastic Bone Metastases.

Bone remodeling is disrupted in the presence of metastases and can present as osteolytic, osteoblastic or a mixture of the two. Established rat models of osteolytic and mixed metastases have been identified changes in structural and tissue-level properties of bone. The aim of this work was to establish a preclinical rat model of osteoblastic metastases and characterize bone quality changes through image-based evaluation. Female athymic rats (n = 22) were inoculated with human breast cancer cells ZR-75-1 and tumor development tracked over 3-4 months with bioluminescence and in-vivo µCT imaging. Bone tissue-level stereological features were quantified on ex-vivo µCT imaging. Histopathology verified the presence of osteoblastic bone. Bone mineral density distribution was assessed via backscattered electron microscopy. Newly formed osteoblastic bone was associated with reduced mineral content and increased heterogeneity leading to an overall degraded bone quality. Characterizing changes in osteoblastic bone properties is relevant to pre-clinical therapeutic testing and treatment planning.

A machine learning model to estimate myocardial stiffness from EDPVR

In-vivo estimation of mechanical properties of the myocardium is essential for patient-specific diagnosis and prognosis of cardiac disease involving myocardial remodeling, including myocardial infarction and heart failure with preserved ejection fraction. Current approaches use time-consuming finite-element (FE) inverse methods that involve reconstructing and meshing the heart geometry, imposing measured loading, and conducting computationally expensive iterative FE simulations. In this paper, we propose a machine learning (ML) model that feasibly and accurately predicts passive myocardial properties directly from select geometric, architectural, and hemodynamic measures, thus bypassing exhaustive steps commonly required in cardiac FE inverse problems. Geometric and fiber-orientation features were chosen to be readily obtainable from standard cardiac imaging protocols. The end-diastolic pressure-volume relationship (EDPVR), which can be obtained using a single-point pressure-volume measurement, was used as a hemodynamic (loading) feature. A comprehensive ML training dataset in the geometry-architecture-loading space was generated, including a wide variety of partially synthesized rodent heart geometry and myofiber helicity possibilities, and a broad range of EDPVRs obtained using forward FE simulations. Latin hypercube sampling was used to create 2500 examples for training, validation, and testing. A multi-layer feed-forward neural network (MFNN) was used as a deep learning agent to train the ML model. The model showed excellent performance in predicting stiffness parameters a_f and b_f associated with fiber direction (R^2_{a_f}=99.471% and R^2_{b_f}=92.837%). After conducting permutation feature importance analysis, the ML performance further improved for b_f (R^2_{b_f}=96.240%), and the left ventricular volume and endocardial area were found to be the most critical geometric features for accurate predictions. The ML model predictions were evaluated further in two cases: (i) rat-specific stiffness data measured using ex-vivo mechanical testing, and (ii) patient-specific estimation using FE inverse modeling. Excellent agreements with ML predictions were found for both cases. The trained ML model offers a feasible technology to estimate patient-specific myocardial properties, thus, bridging the gap between EDPVR, as a confounded organ-level metric for tissue stiffness, and intrinsic tissue-level properties. These properties provide incremental information relative to traditional organ-level indices for cardiac function, improving the clinical assessment and prognosis of cardiac diseases.

Bone material strength index as measured by in vivo impact microindentation is normal in subjects with high-energy trauma fractures

SummaryBone material properties were assessed using impact microindentation in patients with high-energy trauma fractures. Compared to patients with low-energy trauma fractures, bone material strength index was significantly higher in patients with high-energy trauma fractures, and did not differ between patients with osteopenia and those with osteoporosis within each trauma group.IntroductionImpact microindentation (IMI) is a technique to assess tissue-level properties of bone at the tibia. Bone material strength index (BMSi), measured by IMI, is decreased in patients with low-energy trauma fractures, independently of areal bone mineral density (aBMD), but there is no information about BMSi in patients with high-energy trauma fractures. In the present study, we evaluated tissue-level properties of bone with IMI in patients with high-energy trauma fractures.MethodsBMSi was measured 3.0 months (IQR 2.0–5.8) after the fracture in 40 patients with high-energy trauma and 40 age- and gender-matched controls with low-energy trauma fractures using the OsteoProbe® device.ResultsMean age of high- and low-energy trauma patients was 57.7 ± 9.1 and 57.2 ± 7.7 years, respectively (p = 0.78). Fracture types were comparable in high- vs low-energy trauma patients. Lumbar spine (LS)-aBMD, but not femoral neck (FN)-aBMD, was higher in high- than in low-energy trauma patients (LS 0.96 ± 0.13 vs 0.89 ± 0.13 g/cm2, p = 0.02; FN 0.75 ± 0.09 vs 0.72 ± 0.09 g/cm2, p = 0.09). BMSi was significantly higher in high- than in low-energy trauma patients (84.4 ± 5.0 vs 78.0 ± 4.6, p = 0.001), also after adjusting for aBMD (p = 0.003). In addition, BMSi did not differ between patients with osteopenia and those with osteoporosis within each trauma group.ConclusionOur data demonstrate that BMSi and LS-aBMD, but not FN-aBMD, are significantly higher in high-energy trauma patients compared to matched controls with similar fractures from low-energy trauma. Further studies of non-osteoporotic patients with high-energy trauma fracture with measurements of BMSi are warranted to determine whether IMI might help in identifying those with reduced bone strength.

Assessing bone maturity: Compositional and mechanical properties of rib cortical bone at different ages.

Understanding what maturity entails for bone, when it arrives, and its pre- and post-maturity traits and properties are very important for understanding its evolution and physiology. There is a clear but fine distinction between the chronological age of bone (the age of its donor) and the tissue age of the bone packets it comprises at the microscopic level. Whole bone fragility changes with age due to mass and architecture effects, but so do the properties of bone at the tissue level. Tissue age and tissue-level properties are therefore increasingly attracting a great deal of attention recently. The present study investigated compositional and material changes in the hydroxyapatite crystals, the collagenous phase, changes in bone matrix composition and its nanoindentation properties and their decline with chronological age in later life. The aim was to track the age threshold at which cortical bone arrives at maturity and what happens following that threshold. To do so FTIR, DSC/TGA, XRD, nanoindentation and microindentation were used to investigate rib cortical bone material across a cohort of 86 individuals from one ethnic group with age spanning between 17 and 82 years. Results of this cross-sectional study showed a clear increase in mineral content relative to the organic and water contents across all ages. Furthermore, an increase in crystal size and consequent decrease in strain (coherence length) was detected associated with secondary mineralisation and an increase in carbonate substitution. Overall, we observe a number of modifications which contribute to a typical functional behaviour of bone showing an increase in both indentation modulus and hardness until the age of about 35 after which both of these properties decline gradually and concomitantly to other physicochemical changes and seemingly until the end of one's life.

Diaphragm muscle fibrosis involves changes in collagen organization with mechanical implications in Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), diaphragm muscle dysfunction results in respiratory insufficiency, a leading cause of death in patients. Increased muscle stiffness occurs with buildup of fibrotic tissue, characterized by excessive accumulation of extracellular matrix (ECM) components such as collagen, and prevents the diaphragm from achieving the excursion lengths required for respiration. However, changes in mechanical properties are not explained by collagen amount alone and we must consider the complex structure and mechanics of fibrotic tissue. The goals of our study were to 1) determine if and how collagen organization changes with the progression of DMD in diaphragm muscle tissue and 2) predict how collagen organization influences the mechanical properties of the ECM. We first visualized collagen structure with scanning electron microscopy (SEM) images and then developed an analysis framework to quantify collagen organization and generate image-based finite-element models. Image analysis revealed increased collagen fiber straightness and alignment in mdx over wild type (WT) at 3 mo (straightness: mdx = 0.976 ± 0.0108, WT = 0.887 ± 0.0309, alignment: mdx = 0.876 ± 0.0333, WT = 0.759 ± 0.0416) and 6 mo (straightness: mdx = 0.942 ± 0.0182, WT = 0.881 ± 0.0163, alignment: mdx = 0.840 ± 0.0315, WT = 0.759 ± 0.0368). Collagen fibers retained a transverse orientation relative to muscle fibers (70°-90°) in all groups. Mechanical models predicted an increase in the transverse relative to longitudinal (muscle fiber direction) stiffness, with stiffness ratio (transverse/longitudinal) increased in mdx over WT at 3 mo (mdx = 5.45 ± 2.04, WT = 1.97 ± 0.670) and 6 mo (mdx = 4.05 ± 0.985, WT = 1.96 ± 0.506). This study revealed changes in diaphragm ECM structure and mechanics during disease progression in the mdx muscular dystrophy mouse phenotype, highlighting the need to consider the role of collagen organization on diaphragm muscle function.NEW & NOTEWORTHY Scanning electron microscopy images of decellularized diaphragm muscle from WT and mdx, Duchenne muscular dystrophy model, mice revealed that collagen fibers in the epimysium are oriented transverse to muscle fibers, with age- and disease-dependent changes in collagen arrangement. Finite-element models generated from these images predicted that changes in collagen arrangement during disease progression influence the mechanical properties of the extracellular matrix. Thus, changes in collagen fiber-level structure are implicated on tissue-level properties during fibrosis.

Attachment of cartilage wear particles to the synovium negatively impacts friction properties

Cartilage wear particles are released into the synovial fluid by mechanical and chemical degradation of the articular surfaces during osteoarthritis and attach to the synovial membrane. Accumulation of wear particles could alter key tissue-level mechanical properties of the synovium, hindering its characteristically low-friction interactions with underlying articular surfaces in the synovial joint. The present study employs a custom loading device to further the characterization of native synovium friction properties, while investigating the hypothesis that attachment of cartilage wear particles increases friction coefficient. Juvenile bovine synovium demonstrated characteristically low friction coefficients in sliding contact with glass, in agreement with historical measurements. Friction coefficient increased with higher normal load in saline, while lubrication with native synovial fluid maintained low friction coefficients at higher loads. Cartilage wear particles generated from juvenile bovine cartilage attached directly to synovium explants in static culture, with incorporation onto the tissue denoted by cell migration onto the particle surface. In dilute synovial fluid mimicking the decreased lubricating properties during osteoarthritis, wear particle attachment significantly increased friction coefficient against glass, and native cartilage and synovium. In addition to providing a novel characterization of synovial joint tribology this work highlights a potential mechanism for cartilage wear particles to perpetuate the degradative environment of osteoarthritis by modulating tissue-level properties of the synovium that could impact macroscopic wear as well as mechanical stimuli transmitted to resident cells.

Microstructure Determines Apparent-Level Mechanics Despite Tissue-Level Anisotropy and Heterogeneity of Individual Plates and Rods in Normal Human Trabecular Bone.

Trabecular plates and rods determine apparent elastic modulus and yield strength of trabecular bone, serving as important indicators of bone's mechanical integrity in health and disease. Although trabecular bone's apparent-level mechanical properties have been widely reported, tissue mechanical properties of individual trabeculae have not been fully characterized. We systematically measured tissue mineral density (TMD)-dependent elastic modulus of individual trabeculae using microindentation and characterized its anisotropy as a function of trabecular type (plate or rod), trabecular orientation in the global coordinate (longitudinal, oblique, or transverse along the anatomic loading axis), and indentation direction along the local trabecular coordinate (axial or lateral). Human trabecular bone samples were scanned by micro-computed tomography for TMD and microstructural measurements. Individual trabecula segmentation was used to decompose trabecular network into individual trabeculae, where trabecular type and orientation were determined. We performed precise, selective indentation of trabeculae in each category using a custom-built, microscope-coupled microindentation device. Co-localization of TMD at each indentation site was performed to obtain TMD-to-modulus correlations. We found significantly higher TMD and tissue modulus in trabecular plates than rods. Regardless of trabecular type and orientation, axial tissue modulus was consistently higher than lateral tissue modulus, with ratios ranging from 1.13 to 1.41. Correlations between TMD and tissue modulus measured from axial and lateral indentations were strong but distinct: axial correlation predicted higher tissue modulus than lateral correlation at the same TMD level. To assess the contribution of experimentally measured anisotropic tissue properties of individual trabeculae to apparent-level mechanics, we constructed non-linear micro-finite element models using a new set of trabecular bone samples and compared model predictions to mechanical testing measurements. Heterogeneous anisotropic models accurately predicted apparent elastic modulus but were no better than a simple homogeneous isotropic model. Variances in tissue-level properties may therefore contribute nominally to apparent-level mechanics in normal human trabecular bone. © 2021 American Society for Bone and Mineral Research (ASBMR).

Effects of Scaffold Architecture, Materials, and Loading on Cellular Micromechanical Environment in Tissue Engineering Strategies

In tissue engineering (TE) strategies, cell processes are regulated by mechanical stimuli. Although TE scaffolds have been developed to replicate tissue-level mechanical properties, it is experimentally prohibitive to measure and prescribe the cellular micromechanical environment (CME) generated within these constructs. Accordingly, this study aimed to fill this lack of understanding by modelling the CME in TE scaffolds using the finite element method. A repeating unit of composite fiber scaffold for annulus fibrosus repair with a fibrin hydrogel matrix was prescribed a series of loading, material, and architectural parameters. The CME was predicted and the corresponding cell phenotypes were predicted based on previously hypothesized criteria. Scaffold multi-axial loading was demonstrated as the most pertinent parameter contributing to the CME criteria being satisfied. Specifically, radial-direction compression with biaxial tension lead to a prediction of regeneration for 66.5% of the cell volumes. Additionally, the architectural scale had a moderate influence on the CME with minimal change in the tissue-level properties of the scaffold. All other scaffold materials and architectures considered had secondary influences on the predicted regeneration by modifying the scaffold loading. By predicting the regeneration potential of different scaffold designs, the developed high-fidelity computational tool described in this study enables for a more comprehensive understanding of the relationship between tissue-level and cell-level mechanics for a broad range of tissue engineering applications.

Estimating tissue-level properties of porcine talar subchondral bone

Tissue-level properties of bone play an important role when characterising apparent-level bone biomechanical behaviour and yet little is known about its effect at this hierarchical level. In combination with trabecular morphological data these properties can be used to predict bone strength, which becomes an invaluable tool for clinicians in patient treatment planning.This study developed specimen-specific micro-finite element (μFE) models using validated continuum-level models, containing grayscale-derived material properties, to indirectly establish tissue-level properties of porcine talar subchondral bone.Specimen-specific continuum finite element (hFE) models of subchondral trabecular bone were setup using μCT data of ten cylindrical specimens extracted from juvenile porcine tali. The models were validated using quasi-static uniaxial compression testing. Validated hFE models were used to calibrate the tissue modulus of corresponding μFE models by minimising the difference between the μFE and hFE stiffness values. Key trabecular morphological indices (BV/TV, DA, Conn.D, Tb.Th, EF) were evaluated.Good agreement was observed between hFE models and experiment (CCC = 0.66). Calibrated Etiss was 504 ± 37.65 MPa. Average BV/TV and DA for μFE specimens were 0.37 ± 0.05 and 0.68 ± 0.11, respectively. BV/TV (r2 = 0.667) correlated highly with μFE stiffness.The small intra-specimen variation to tissue-level properties suggests that variations to apparent-level stiffness originate from variations to microarchitecture rather than tissue mechanical properties.

Historical Review of Combined Experimental and Computational Approaches for Investigating Annulus Fibrosus Mechanics.

Intervertebral disc research has sought to develop a deeper understanding of spine biomechanics, the complex relationship between disc health and back pain, and the mechanisms of spinal injury and repair. To do so, many researchers have focused on characterizing tissue-level properties of the disc, where the roles of tissue subcomponents can be more systematically investigated. Unfortunately, experimental challenges often limit the ability to measure important disc tissue- and subtissue-level behaviors, including fiber-matrix interactions, transient nutrient and electrolyte transport, and damage propagation. Numerous theoretical and numerical modeling frameworks have been introduced to explain, complement, guide, and optimize experimental research efforts. The synergy of experimental and computational work has significantly advanced the field, and these two aspects have continued to develop independently and jointly. Meanwhile, the relationship between experimental and computational work has become increasingly complex and interdependent. This has made it difficult to interpret and compare results between experimental and computational studies, as well as between solely computational studies. This paper seeks to explore issues of model translatability, robustness, and efficient study design, and to propose and motivate potential future directions for experimental, computational, and combined tissue-level investigations of the intervertebral disc.

Impact microindentation assesses subperiosteal bone material properties in humans.

Impact microindentation (IMI) is a Reference Point Indentation technique measuring tissue-level properties of cortical bone in humans in vivo. The nature, however, of the properties that can affect bone strength is incompletely understood. In the present study we examined bone material properties in transiliac bone biopsies obtained concurrently with measurements of Bone Material Strength index (BMSi) by IMI in 12 patients with different skeletal disorders and a wide range of BMD, with or without fractures (8 males, 4 females, mean age 48±12.2 (SD) years, range 15-60 years). IMI was performed in the mid-shaft of the right tibia with a hand-held microindenter (OsteoProbe). Cancellous and cortical bone mineralization density distributions (BMDD) were measured in the entire biopsy bone area by quantitative backscattered electron imaging. Raman measurements were obtained right at the outer edge of the cortex, and 5, 50, 100, 500μm inwards. The calculated parameters were: i) Mineral and organic matrix content as well as the mineral / matrix ratio. ii) Nanoporosity. iii) Glycosaminoglycan content. iv) Pyridinoline content. v) Maturity/crystallinity of the apatite crystallites. There was no relationship between BMSi values with any measurement of mineral content of whole bone tissue (BMD, BMDD) or maturity/crystallinity of bone mineral. On the other hand, a positive correlation between BMSi and local mineral content, and an inverse correlation between BMSi and nanoporosity at the mineralized subperiosteal edge of the sample and at 5μm inwards was found. A positive correlation was also observed between BMSi and pyridinoline content at the same locations. These results indicate that local mineral content, nanoporosity and pyridinoline content at the subperiosteal site in the transiliac bone biopsy are linked to the BMSi values measured in the tibia. As both high porosity at the nano level and low pyridinoline content of the bone matrix can negatively impact bone strength, our findings suggest that BMSi most likely assesses subperiosteal bone material properties.

Insights Into the Pathogenesis of Catecholaminergic Polymorphic Ventricular Tachycardia From Engineered Human Heart Tissue.

Modeling of human arrhythmias with induced pluripotent stem cell-derived cardiomyocytes has focused on single-cell phenotypes. However, arrhythmias are the emergent properties of cells assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported. Here, we report an optogenetically based, human engineered tissue model of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia caused by mutation of the cardiac ryanodine channel and triggered by exercise. We developed a human induced pluripotent stem cell-derived cardiomyocyte-based platform to study the tissue-level properties of engineered human myocardium. We investigated pathogenic mechanisms in CPVT by combining this novel platform with genome editing. In our model, CPVT tissues were vulnerable to developing reentrant rhythms when stimulated by rapid pacing and catecholamine, recapitulating hallmark features of the disease. These conditions elevated diastolic Ca2+ levels and increased temporal and spatial dispersion of Ca2+ wave speed, creating a vulnerable arrhythmia substrate. Using Cas9 genome editing, we pinpointed a single catecholamine-driven phosphorylation event, ryanodine receptor-serine 2814 phosphorylation by Ca2+/calmodulin-dependent protein kinase II, that is required to unmask the arrhythmic potential of CPVT tissues. Our study illuminates the molecular and cellular pathogenesis of CPVT and reveals a critical role of calmodulin-dependent protein kinase II-dependent reentry in the tissue-scale mechanism of this disease. We anticipate that this approach will be useful for modeling other inherited and acquired cardiac arrhythmias.

Osteocytes and mechanical loading: The Wnt connection.

Bone adapts to the mechanical forces that it experiences. Orthodontic tooth movement harnesses the cell- and tissue-level properties of mechanotransduction to achieve alignment and reorganization of the dentition. However, the mechanisms of action that permit bone resorption and formation in response to loads placed on the teeth are incompletely elucidated, though several mechanisms have been identified. Wnt/Lrp5 signalling in osteocytes is a key pathway that modulates bone tissue's response to load. Numerous mouse models that harbour knock-in, knockout and transgenic/overexpression alleles targeting genes related to Wnt signalling point to the necessity of Wnt/Lrp5, and its localization to osteocytes, for proper mechanotransduction in bone. Alveolar bone is rich in osteocytes and is a highly mechanoresponsive tissue in which components of the canonical Wnt signalling cascade have been identified. As Wnt-based agents become clinically available in the next several years, the major challenge that lies ahead will be to gain a more complete understanding of Wnt biology in alveolar bone so that improved/expedited tooth movement becomes a possibility.

Loss of the PTH/PTHrP receptor along the osteoblast lineage limits the anabolic response to exercise.

Exercise and physical activity are critical to maintain bone mass and strength throughout life. Both exercise and physical activity subject bone to a unique combination of stimuli in the forms of dynamic loading and a systemic increase in parathyroid hormone (PTH). Although dynamic loading is considered to be the primary osteogenic stimuli, the influence of increasing PTH levels remains unclear. We hypothesize that activation of the PTH/PTH-related peptide type 1 receptor (PPR) along the osteoblast lineage facilitates bone formation and improved mechanical properties in response to exercise. To test this hypothesis, conditional PPR-knockout mice (PPRcKO) were generated in which PPR expression was deleted along the osteoblast lineage under the osterix promoter. At 8-weeks of age, both PPRfl/fl and PPRcKO mice were subjected to treadmill running or sedentary conditions for 5-weeks. Under sedentary conditions, PPRcKO mice displayed significantly less bone mass as well as smaller structural-level strength (yield-load and ultimate load), while tissue level properties were largely unaffected. However, PPRcKO mice exposed to exercise displayed significantly less structural-level and tissue-level mechanical properties when compared to exercised PPRfl/fl mice. Overall, these data demonstrate that PPR expression along the osteoblast lineage is essential for exercise to improve the mechanical properties of cortical bone. Furthermore, the influence of PPR activation on material properties is unique to exercise and not during normal growth and development.

Mechanical behavior of metastatic vertebrae are influenced by tissue architecture, mineral content, and organic feature alterations.

Diminished vertebral mechanical behavior with metastatic involvement is typically attributed to modified architecture and trabecular bone content. Previous work has identified organic and mineral phase bone quality changes in the presence of metastases, yet limited work exists on the potential influence of such tissue level modifications on vertebral mechanical characteristics. This work seeks to determine correlations between features of bone (structural and tissue level) and mechanical behavior in metastatically involved vertebral bone. It is hypothesized that tissue level properties (mineral and organic) will improve these correlations beyond architectural properties and BMD alone. Twenty-four female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic (N = 8) or mixed (osteolytic/osteoblastic, N = 7) metastases, respectively. Twenty-one days post-inoculation L1-L3 pathologic vertebral motion segments were excised and μCT imaged. 3D morphometric parameters and axial rigidity of the L2 vertebrae were quantified. Sequential loading and μCT imaging measured progression of failure, stiffness and peak force. Relationships between mechanical testing (whole bone and tissue-level) and tissue-level material property modifications with metastatic involvement were evaluated utilizing linear regression models. Osteolytic involvement reduced vertebral trabecular bone volume, structure, CT-derived axial rigidity, stiffness and failure force compared to healthy controls (N = 9). Mixed metastases demonstrated similar trends. Previously assessed collagen cross-linking and proline-based residues were correlated to mechanical behavior and improved the predictive ability of the regression models. Similarly, collagen organization improved predictive regression models for metastatic bone hardness. This work highlights the importance of both bone content/architecture and organic tissue-level features in characterizing metastatic vertebral mechanics. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3013-3022, 2018.