Osteochondral fluid transport in an ex vivo system.

OBJECTIVE: Alterations to bone-to-cartilage fluid transport may contribute to the development of osteoarthritis (OA). Larger biological molecules in bone may transport from bone-to-cartilage (e.g., insulin, 5 kDa). However, many questions remain about fluid transport between these tissues. The objectives of this study were to (1) test for diffusion of 3 kDa molecular tracers from bone-to-cartilage and (2) assess potential differences in bone-to-cartilage fluid transport between different loading conditions. DESIGN: Osteochondral cores extracted from bovine femurs (N = 10 femurs, 10 cores/femur) were subjected to either no-load (i.e., pure diffusion), pre-load only, or cyclic compression (5 ± 2% or 10 ± 2% strain) in a two-chamber bioreactor. The bone was placed into the bone compartment followed by a 3 kDa dextran tracer, and tracer concentrations in the cartilage compartment were measured every 5 min for 120 min. Tracer concentrations were analyzed for differences in beginning, peak, and equilibrium concentrations, loading effects, and time-to-peak tracer concentration. RESULTS: Peak tracer concentration in the cartilage compartment was significantly higher compared to the beginning and equilibrium tracer concentrations. Cartilage-compartment tracer concentration and maximum fluorescent intensity were influenced by strain magnitude. No time-to-peak relationship was found between strain magnitudes and cartilage-compartment tracer concentration. CONCLUSION: This study shows that bone-to-cartilage fluid transport occurs with 3 kDa dextran molecules. These are larger molecules to move between bone and cartilage than previously reported. Further, these results demonstrate the potential impact of cyclic compression on osteochondral fluid transport. Determining the baseline osteochondral fluid transport in healthy tissues is crucial to elucidating the mechanisms OA pathology.

Rapamycin does not alter bone microarchitecture or material properties quality in young-adult and aged female C57BL/6 mice.

Advancing age is the strongest risk factor for osteoporosis and skeletal fragility. Rapamycin is an FDA-approved immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) complex, extends lifespan, and protects against aging-related diseases in multiple species; however, the impact of rapamycin on skeletal tissue is incompletely understood. We evaluated the effects of a short-term, low-dosage, interval rapamycin treatment on bone microarchitecture and strength in young-adult (3 mo old) and aged female (20 mo old) C57BL/6 mice. Rapamycin (2 mg/kg body mass) was administered via intraperitoneal injection 1×/5 d for a duration of 8 wk; this treatment regimen has been shown to induce geroprotective effects while minimizing the side effects associated with higher rapamycin dosages and/or more frequent or prolonged delivery schedules. Aged femurs exhibited lower cancellous bone mineral density, volume, trabecular connectivity density and number, higher trabecular thickness and spacing, and lower cortical thickness compared to young-adult mice. Rapamycin had no impact on assessed microCT parameters. Flexural testing of the femur revealed that both yield strength and ultimate strength were lower in aged mice compared to young-adult mice. There were no effects of rapamycin on these or other measures of bone biomechanics. Age, but not rapamycin, altered local and global measures of bone turnover. These data demonstrate that short-term, low-dosage interval rapamycin treatment does not negatively or positively impact the skeleton of young-adult and aged mice.

Sex-specific effects of Fat-1 transgene on bone material properties, size, and shape in mice.

Western diets are becoming increasingly common around the world. Western diets have high omega 6 (ω-6) and omega 3 (ω-3) fatty acids and are linked to bone loss in humans and animals. Dietary fats are not created equal; therefore, it is vital to understand the effects of specific dietary fats on bone. We aimed to determine how altering the endogenous ratios of ω-6:ω-3 fatty acids impacts bone accrual, strength, and fracture toughness. To accomplish this, we used the Fat-1 transgenic mice, which carry a gene responsible for encoding a ω-3 fatty acid desaturase that converts ω-6 to ω-3 fatty acids. Male and female Fat-1 positive mice (Fat-1) and Fat-1 negative littermates (WT) were given either a high-fat diet (HFD) or low-fat diet (LFD) at 4 wk of age for 16 wk. The Fat-1 transgene reduced fracture toughness in males. Additionally, male BMD, measured from DXA, decreased over the diet duration for HFD mice. In males, neither HFD feeding nor the presence of the Fat-1 transgene impacted cortical geometry, trabecular architecture, or whole-bone flexural properties, as detected by main group effects. In females, Fat-1-LFD mice experienced increases in BMD compared to WT-LFD mice; however, cortical area, distal femur trabecular thickness, and cortical stiffness were reduced in Fat-1 mice compared to pooled WT controls. However, reductions in stiffness were caused by a decrease in bone size and were not driven by changes in material properties. Together, these results demonstrate that the endogenous ω-6:ω-3 fatty acid ratio influences bone material properties in a sex-dependent manner. In addition, Fat-1 mediated fatty acid conversion was not able to mitigate the adverse effects of HFD on bone strength and accrual.

Aging alters the subchondral bone response 7 days after noninvasive traumatic joint injury in C57BL/6JN mice.

Posttraumatic osteoarthritis (PTOA) commonly develops following anterior cruciate ligament (ACL) injuries, affecting around 50% of individuals within 10-20 years. Recent studies have highlighted early changes in subchondral bone structure after ACL injury in adolescent or young adult mice, which could contribute to the development of PTOA. However, ACL injuries do not only occur early in life. Middle-aged and older patients also experience ACL injuries and PTOA, but whether the aged subchondral bone also responds rapidly to injury is unknown. This study utilized a noninvasive, single overload mouse injury model to assess subchondral bone microarchitecture, turnover, and material properties in both young adults (5 months) and early old age (22 months) female C57BL/6JN mice at 7 days after injury. Mice underwent either joint injury (i.e., produces ACL tears) or sham injury procedures on both the loaded and contralateral limbs, allowing evaluation of the impacts of injury versus loading. The subchondral bone response to ACL injury is distinct for young adult and aged mice. While 5-month mice show subchondral bone loss and increased bone resorption postinjury, 22-month mice did not show loss of bone structure and had lower bone resorption. Subchondral bone plate modulus increased with age, but not with injury. Both ages of mice showed several bone measures were altered in the contralateral limb, demonstrating the systemic skeletal response to joint injury. These data motivate further investigation to discern how osteochondral tissues differently respond to injury in aging, such that diagnostics and treatments can be refined for these demographics.

Aging decreases osteocyte lacunar-canalicular turnover in female C57BL/6 mice.

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in LCS turnover and how these processes change with aging. Furthermore, whether LCS turnover depends on tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) femurs from young (5 mo) and early-old-age (22 mo) female C57BL/6JN mice. LCS bone mineralization was measured by the presence of perilacunar fluorochrome labels. LCS bone resorption was measured by immunohistochemical markers of bone resorption. The dynamics of LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 days and 16 days before euthanasia. Osteocyte participation in mineralizing their surroundings is highly abundant in both cortical and cancellous bone of young adult mice but significantly decreases with aging. LCS bone resorption also decreases with aging. Aging has a greater impact on LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent LCS turnover have larger lacunae in both age groups. The impacts of aging on LCS turnover also varies with cortical region of interest and intracortical location, suggesting a dependence on tissue strain. The impact of aging on decreasing LCS turnover may have significant implications for bone quality and mechanosensation.

Germ-Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance.

The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ-free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20- to 21-week-old) C57BL/6J GF and conventionally raised female and male mice (n = 6-10/group). Trabecular microarchitecture and cortical geometry were measured from micro-CT of the femur distal metaphysis and cortical midshaft. Whole-femur strength and estimated material properties were measured using three-point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back-scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole-bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The flying insect thoracic cuticle is heterogenous in structure and in thickness-dependent modulus gradation.

The thorax is a specialized structure central to insect flight. In the thorax, flight muscles are surrounded by a thin layer of cuticle. The structure, composition, and material properties of this chitinous structure may influence the efficiency of the thorax in flight. However, these properties, as well as their variation throughout the thorax and between insect taxa, are not known. We provide a multi-faceted assessment of thorax cuticle for fliers with asynchronous (honey bee; Apis mellifera) and synchronous (hawkmoth; Manduca sexta) muscles. These muscle types are defined by the relationship between their activation frequency and the insect's wingbeat frequency. We investigated cuticle structure using histology, resilin distribution through confocal laser scanning microscopy, and modulus gradation with nanoindentation. Our results suggest that thorax cuticle properties are highly dependent on anatomical region and species. Modulus gradation, but not mean modulus, differed between the two types of fliers. In some regions, A. mellifera had a positive linear modulus gradient from cuticle interior to exterior of about 2 GPa. In M. sexta, modulus values through cuticle thickness were not well represented by linear fits. We utilized finite element modeling to assess how measured modulus gradients influenced maximum stress in cuticle. Stress was reduced when cuticle with a linear gradient was compressed from the high modulus side. These results support the protective role of the A. mellifera thorax cuticle. Our multi-faceted assessment advances our understanding of thorax cuticle structural and material heterogeneity and the potential benefits of material gradation to flying insects. STATEMENT OF SIGNIFICANCE: The insect thorax is essential for efficient flight but questions remain about the contribution of the exoskeletal cuticle. We investigated the microscale properties of the thorax cuticle, a crucial step to determine its role in flight. Techniques including histology, nanoindentation, and confocal laser scanning microscopy revealed that cuticle properties vary through cuticle thickness, by thorax region, and between species with asynchronous (honey bee; Apis mellifera) and synchronous (hawkmoth; Manduca sexta) muscles. This variation highlights the importance of high resolution cuticle assessment for flying insect lineages and points to factors that may (modulus gradation) and may not (mean modulus) contribute to different flight forms. Understanding material variation in the thorax may inform design of technologies inspired by insects, such as mobile micro robots.

Perilacunar bone tissue exhibits sub-micrometer modulus gradation which depends on the recency of osteocyte bone formation in both young adult and early-old-age female C57Bl/6 mice.

Osteocytes resorb and replace bone local to the lacunar-canalicular system (LCS). However, whether osteocyte remodeling impacts bone quality adjacent to the LCS is not understood. Further, while aging is well-established to decrease osteocyte viability and truncate LCS geometry, it is unclear if aging also decreases perilacunar bone quality. To address these questions, we employed atomic force microscopy (AFM) to generate nanoscale-resolution modulus maps for cortical femur osteocyte lacunae from young (5-month) and early-old-age (22-month) female C57Bl/6 mice. AFM-mapped lacunae were also imaged with confocal laser scanning microscopy to determine which osteocytes recently deposited bone as determined by the presence of fluorochrome labels administered 2d and 8d before euthanasia. Modulus gradation with distance from the lacunar wall was compared for labeled (i.e., bone forming) and non-labeled lacunae in both young and aged mice. All mapped lacunae showed sub-microscale modulus gradation, with peak modulus values 200-400 nm from the lacunar wall. Perilacunar modulus gradations depended on the recency of osteocyte bone formation (i.e., the presence of labels). For both ages, 2d-labeled perilacunar bone had lower peak and bulk modulus compared to non-labeled perilacunar bone. Lacunar length reduced with age, but lacunar shape and size were not strong predictors of modulus gradation. Our findings demonstrate for the first time that osteocyte perilacunar remodeling impacts bone tissue modulus, one contributor to bone quality. Given the immense scale of the LCS, differences in perilacunar modulus resulting from osteocyte remodeling activity may affect the quality of a substantial amount of bone tissue.

The Cortical Bone Metabolome of C57BL/6J Mice Is Sexually Dimorphic.

Cortical bone quality, which is sexually dimorphic, depends on bone turnover and therefore on the activities of remodeling bone cells. However, sex differences in cortical bone metabolism are not yet defined. Adding to the uncertainty about cortical bone metabolism, the metabolomes of whole bone, isolated cortical bone without marrow, and bone marrow have not been compared. We hypothesized that the metabolome of isolated cortical bone would be distinct from that of bone marrow and would reveal sex differences. Metabolite profiles from liquid chromatography-mass spectrometry (LC-MS) of whole bone, isolated cortical bone, and bone marrow were generated from humeri from 20-week-old female C57Bl/6J mice. The cortical bone metabolomes were then compared for 20-week-old female and male C57Bl/6J mice. Femurs from male and female mice were evaluated for flexural material properties and were then categorized into bone strength groups. The metabolome of isolated cortical bone was distinct from both whole bone and bone marrow. We also found sex differences in the isolated cortical bone metabolome. Based on metabolite pathway analysis, females had higher lipid metabolism, and males had higher amino acid metabolism. High-strength bones, regardless of sex, had greater tryptophan and purine metabolism. For males, high-strength bones had upregulated nucleotide metabolism, whereas lower-strength bones had greater pentose phosphate pathway metabolism. Because the higher-strength groups (females compared with males, high-strength males compared with lower-strength males) had higher serum type I collagen cross-linked C-telopeptide (CTX1)/procollagen type 1 N propeptide (P1NP), we estimate that the metabolomic signature of bone strength in our study at least partially reflects differences in bone turnover. These data provide novel insight into bone bioenergetics and the sexual dimorphic nature of bone material properties in C57Bl/6 mice. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Development and analytical validation of a finite element model of fluid transport through osteochondral tissue.

Fluid transport is critical to joint health. In this study we evaluate an unexplored component of joint fluid transport -fluid transport between cartilage and bone. Such transport across the cartilage-bone interface could potentially provide chondrocytes with an additional source of nutrients and signaling molecules. A biphasic viscoelastic model using an ellipsoidal fiber distribution was created with three distinct layers of cartilage (superficial zone, middle zone, and deep zone) along with a layer of subchondral bone. For stress-relaxation in unconfined compression, our results for compressive stress, radial stress, and effective fluid pressure were compared with established biphasic analytical solutions. Our model also shows the development of fluid pressure gradients at the cartilage-bone interface during loading. Fluid pressure gradients that develop at the cartilage-bone interface show consistently higher pressures in cartilage following the initial loading to 10% stain, followed by convergence of the pressures in cartilage and bone during the 400 s relaxation period. These results provide additional evidence that fluid is transported between cartilage and bone during loading and improves upon estimates of the magnitude of this effect through incorporating a realistic distribution and estimate of the collagen ultrastructure. Understanding fluid transport between cartilage and bone may be key to new insights about the mechanical and biological environment of both tissues in health and disease.

Sex differences in the effect of diet, obesity, and exercise on bone quality and fracture toughness.

Bone fragility and obesity are both diseases that are multifactorial in etiology and pathology. The contributing role of high fat diet (HFD) versus energy overconsumption on bone health is controversial. Exercise is often prescribed for improving bone health, but it is unclear whether HFD or overconsumption influences skeletal adaptations to exercise. Female and male Wistar rats were fed HFD or low fat diet (LFD) for 10 weeks, starting at 8 weeks of age. Within HFD, rats were labeled Obesity-Resistant (OR) or Obesity-Prone (OP) based on weight and fat gain. Within each diet and phenotype group, rats were randomized to treadmill exercise or sedentary control (SED) for the final 4 weeks. Femurs were assessed for fracture toughness. Cortical lamellar and nonlamellar bone microscale material behavior and chemistry were assessed using nanoindentation and Raman spectroscopy. Female bones had higher fracture toughness and mineral: matrix ratio than male bones. Diet and energy overconsumption affected bone characteristics in a sex-dependent manner, where the divergence between OP and OR in response to HFD occurred more rapidly in males. Diet composition, in general, had a stronger effect on bone quality than overconsumption. HFD dramatically decreased bone size and lamellar mineral:matrix compared to LFD. Effects of short-term exercise training on microscale tissue properties were generally more robust with LFD. Exercise enhanced the contrast between lamellar and nonlamellar bone for nanoindentation modulus but decreased this contrast for plastic work. Our data demonstrate the complexities in the relationship between diet and obesity and highlight the importance of addressing both aspects when characterizing bone quality and fracture resistance.

Ice-Binding Protein from Shewanella frigidimarinas Inhibits Ice Crystal Growth in Highly Alkaline Solutions.

The ability of a natural ice-binding protein from Shewanella frigidimarina (SfIBP) to inhibit ice crystal growth in highly alkaline solutions with increasing pH and ionic strength was investigated in this work. The purity of isolated SfIBP was first confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography with an ultraviolet detector (SEC-UV). Protein stability was evaluated in the alkaline solutions using circular dichroism spectroscopy, SEC-UV, and SDS-PAGE. SfIBP ice recrystallization inhibition (IRI) activity, a measure of ice crystal growth inhibition, was assessed using a modified splat assay. Statistical analysis of results substantiated that, despite partial denaturation and misfolding, SfIBP limited ice crystal growth in alkaline solutions (pH ≤ 12.7) with ionic strength I ≤ 0.05 mol/L, but did not exhibit IRI activity in alkaline solutions where pH ≥ 13.2 and I ≥ 0.16 mol/L. IRI activity of SfIBP in solutions with pH ≤ 12.7 and I ≤ 0.05 mol/L demonstrated up to ≈ 66% reduction in ice crystal size compared to neat solutions.

Engineered Ureolytic Microorganisms Can Tailor the Morphology and Nanomechanical Properties of Microbial-Precipitated Calcium Carbonate.

We demonstrate for the first time that the morphology and nanomechanical properties of calcium carbonate (CaCO3) can be tailored by modulating the precipitation kinetics of ureolytic microorganisms through genetic engineering. Many engineering applications employ microorganisms to produce CaCO3. However, control over bacterial calcite morphology and material properties has not been demonstrated. We hypothesized that microorganisms genetically engineered for low urease activity would achieve larger calcite crystals with higher moduli. We compared precipitation kinetics, morphology, and nanomechanical properties for biogenic CaCO3 produced by two Escherichia coli (E. coli) strains that were engineered to display either high or low urease activity and the native producer Sporosarcina pasteurii. While all three microorganisms produced calcite, lower urease activity was associated with both slower initial calcium depletion rate and increased average calcite crystal size. Both calcite crystal size and nanoindentation moduli were also significantly higher for the low-urease activity E. coli compared with the high-urease activity E. coli. The relative resistance to inelastic deformation, measured via the ratio of nanoindentation hardness to modulus, was similar across microorganisms. These findings may enable design of novel advanced engineering materials where modulus is tailored to the application while resistance to irreversible deformation is not compromised.

Chronic kidney disease and aging differentially diminish bone material and microarchitecture in C57Bl/6 mice.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.

A new open-source tool for measuring 3D osteocyte lacunar geometries from confocal laser scanning microscopy reveals age-related changes to lacunar size and shape in cortical mouse bone.

Osteocytes can participate in systemic mineral homeostasis through perilacunar maintenance and remodeling, where changes to osteocyte lacunar morphology may affect bone structural integrity, tissue strains, and osteocyte mechanosensitivity. Though aging is associated with both decreased bone quality and altered mineral metabolism, it is not known if osteocyte lacunae undergo age-related changes in geometry. In order to survey lacunar changes with age, we developed an open-source program whereby 3D osteocyte lacunae are automatically segmented and then subsequently reconstructed from confocal laser scanning microscopy (CLSM) depth stacks for quantitative analysis of geometry and orientation. This approach takes advantage of the availability and speed of CLSM while avoiding time-consuming and bias-prone manual segmentation. Unlike conventional approaches used to quantify osteocyte lacunar morphology, CLSM enables facile analysis in three-dimensions with clear identification of osteocyte lacunae. We report that 3D osteocyte lacunae measured by CLSM become smaller, more spherical, more oblate, more spatially disorganized, and more sparsely populated with increased age in C57Bl/6 mouse cortical bone in groups spanning 6-24 months old. Critically, these age-related changes are in large part not observed in 2D analyses from the same samples. These results (1) demonstrate proof-of-concept of an efficient method to quantitatively assess osteocyte lacunae in 3D for application to a wide range of studies and (2) motivate further inquiry into how changes to osteocyte lacunar geometries and perilacunar material contribute to diminished bone quality in aging.

Determination of the True Lateral Grain Size in Organic-Inorganic Halide Perovskite Thin Films.

In this letter, methylammonium lead iodide (MAPbI3) thin films were examined via piezoresponse force microscopy (PFM) and nanoindentation (NI) to determine if long-range atomic order existed across the full width and depth of the apparent grains. From the PFM, the piezoelectric response of the films was strongly correlated with low-index planes of the crystal structure and ferroelastic domains in macroscale solution-grown MAPbI3 crystals, which implied long-range order near the top surface. From the NI, it was found that the induced cracks were straight and extended across the full width of the apparent grains, which indicated that the long-range order was not limited to the near-surface region, but extended through the film thickness. Interestingly, the two MAPbI3 processes examined resulted in subtle differences in the extracted electro-mechanical and fracture properties, but exhibited similar power conversion efficiencies of >17% in completed devices.

Indentation mapping revealed poroelastic, but not viscoelastic, properties spanning native zonal articular cartilage.

Osteoarthrosis is a debilitating disease affecting millions, yet engineering materials for cartilage regeneration has proven difficult because of the complex microstructure of this tissue. Articular cartilage, like many biological tissues, produces a time-dependent response to mechanical load that is critical to cell's physiological function in part due to solid and fluid phase interactions and property variations across multiple length scales. Recreating the time-dependent strain and fluid flow may be critical for successfully engineering replacement tissues but thus far has largely been neglected. Here, microindentation is used to accomplish three objectives: (1) quantify a material's time-dependent mechanical response, (2) map material properties at a cellular relevant length scale throughout zonal articular cartilage and (3) elucidate the underlying viscoelastic, poroelastic, and nonlinear poroelastic causes of deformation in articular cartilage. Untreated and trypsin-treated cartilage was sectioned perpendicular to the articular surface and indentation was used to evaluate properties throughout zonal cartilage on the cut surface. The experimental results demonstrated that within all cartilage zones, the mechanical response was well represented by a model assuming nonlinear biphasic behavior and did not follow conventional viscoelastic or linear poroelastic models. Additionally, 10% (w/w) agarose was tested and, as anticipated, behaved as a linear poroelastic material. The approach outlined here provides a method, applicable to many tissues and biomaterials, which reveals and quantifies the underlying causes of time-dependent deformation, elucidates key aspects of material structure and function, and that can be used to provide important inputs for computational models and targets for tissue engineering. STATEMENT OF SIGNIFICANCE: Elucidating the time-dependent mechanical behavior of cartilage, and other biological materials, is critical to adequately recapitulate native mechanosensory cues for cells. We used microindentation to map the time-dependent properties of untreated and trypsin treated cartilage throughout each cartilage zone. Unlike conventional approaches that combine viscoelastic and poroelastic behaviors into a single framework, we deconvoluted the mechanical response into separate contributions to time-dependent behavior. Poroelastic effects in all cartilage zones dominated the time-dependent behavior of articular cartilage, and a model that incorporates tension-compression nonlinearity best represented cartilage mechanical behavior. These results can be used to assess the success of regeneration and repair approaches, as design targets for tissue engineering, and for development of accurate computational models.

Multi-Scale Mechanical Behavior of the Li3PS4 Solid-Phase Electrolyte.

The need for smaller, lighter, and longer lasting rechargeable batteries is projected to increase rapidly in the coming years because of high demand for portable electronics and electric vehicles. While traditional Li-ion batteries use liquid-phase electrolytes, these suffer from safety risks and low energy density. Solid-phase electrolytes can avoid these issues by enabling a Li metal anode, but tend to fail during cycling due to Li metal dendrite growth between the electrodes. Because Li dendrite nucleation and growth can be viewed in terms of the mechanical behavior of the battery components, it is critical to understand the mechanical response of candidate electrolyte materials. In this work, we use nanoindentation and bulk acoustic techniques to characterize the mechanical properties of ß-Li3PS4, a promising Li-ion conducting ceramic. We find that the bulk and shear moduli of an 80% dense bulk LPS sample are 10-12 GPa and 5-6 GPa, respectively. Although this value of shear modulus may be too low to prevent Li dendrite propagation, it is likely that there are many other mechanical properties that must be taken into account to fully understand Li dendrite nucleation and growth. Ultimately, this work represents a first step in understanding the relationship between Li3PS4 separator manufacture and its mechanical properties.

Moderate chronic kidney disease impairs bone quality in C57Bl/6J mice.

Chronic kidney disease (CKD) increases bone fracture risk. While the causes of bone fragility in CKD are not clear, the disrupted mineral homeostasis inherent to CKD may cause material quality changes to bone tissue. In this study, 11-week-old male C57Bl/6J mice underwent either 5/6th nephrectomy (5/6 Nx) or sham surgeries. Mice were fed a normal chow diet and euthanized 11weeks post-surgery. Moderate CKD with high bone turnover was established in the 5/6 Nx group as determined through serum chemistry and bone gene expression assays. We compared nanoindentation modulus and mineral volume fraction (assessed through quantitative backscattered scanning electron microscopy) at matched sites in arrays placed on the cortical bone of the tibia mid-diaphysis. Trabecular and cortical bone microarchitecture and whole bone strength were also evaluated. We found that moderate CKD minimally affected bone microarchitecture and did not influence whole bone strength. Meanwhile, bone material quality decreased with CKD; a pattern of altered tissue maturation was observed with 5/6 Nx whereby the newest 60µm of bone tissue adjacent to the periosteal surface had lower indentation modulus and mineral volume fraction than more interior, older bone. The variance of modulus and mineral volume fraction was also altered following 5/6 Nx, implying that tissue-scale heterogeneity may be negatively affected by CKD. The observed lower bone material quality may play a role in the decreased fracture resistance that is clinically associated with human CKD.

An autologous cartilage tissue implant NeoCart for treatment of grade III chondral injury to the distal femur: prospective clinical safety trial at 2 years.

BACKGROUND: The healing potential of damaged articular cartilage is limited. The NeoCart is a tissue-engineered collagen matrix seeded with autogenous chondrocytes designed for the repair of hyaline articular cartilage. HYPOTHESIS: The NeoCart implant is well tolerated in the human knee. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Eight patients (treatment group) with full-thickness cartilage injury were treated with the NeoCart and evaluated prospectively. Autogenous chondrocytes provided by arthroscopic biopsy were seeded into a 3-dimensional type I collagen scaffold. The seeded scaffold was subjected to a tissue-engineering protocol including treatment with a bioreactor. Implantation of the prepared cartilage tissue patch was performed via miniarthrotomy and secured with a collagen bioadhesive. Evaluations through 24 months postoperatively included the subjective International Knee Documentation Committee questionnaire, visual analog scale, range of motion, and cartilage-sensitive magnetic resonance imaging (MRI), including quantitative T2 mapping. RESULTS: Pain scores after NeoCart implantation were significantly lower than baseline at 12 and 24 months after the procedure (P < .05). Improved function and motion were also noted at 24 months. Six patients had 67% to 100% defect fill at 24 months with MRI evaluation. One patient had moderate (33%-66%) defect fill, and another patient had poor (less than 33%) defect fill. Partial stratification of T2 values was observed for 2 patients at 12 months and 4 patients at 24 months. No patients experienced arthrofibrosis or implant hypertrophy. CONCLUSION: Pain was significantly reduced 12 and 24 months after NeoCart treatment. Trends toward improved function and motion were observed 24 months after implantation. The MRI indicated implant stability and peripheral integration, defect fill without overgrowth, progressive maturation, and more organized cartilage formation.