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

Osteocytes engage in bone resorption and mineralization surrounding their expansive lacunar-canalicular system (LCS) through peri-LCS turnover. However, fundamental questions persist about where, when, and how often osteocytes engage in peri-LCS turnover and how these processes change with aging. Furthermore, whether peri-LCS turnover is associated with natural variation in cortical tissue strain remains unexplored. To address these questions, we utilized confocal scanning microscopy, immunohistochemistry, and scanning electron microscopy to characterize osteocyte peri-LCS turnover in the cortical (mid-diaphysis) and cancellous (metaphysis) regions of femurs from young adult (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 marker of bone resorption. The dynamics of peri-LCS turnover were estimated from serial fluorochrome labeling, where each mouse was administered two labels between 2 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 peri-LCS turnover dynamics in cancellous bone than in cortical bone. Lacunae with recent peri-LCS turnover are larger in both age groups. While peri-LCS turnover is associated with variation in tissue strain between cortical quadrants and intracortical location for 22 mo mice, these associations were not seen for 5 mo mice. The impact of aging on decreasing peri-LCS turnover may have significant implications for bone quality and mechanosensation.

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.

Biotrapping Ureolytic Bacteria on Sand to Improve the Efficiency of Biocementation.

Microbially induced calcium carbonate precipitation (MICP) has emerged as a novel technology with the potential to produce building materials through lower-temperature processes. The formation of calcium carbonate bridges in MICP allows the biocementation of aggregate particles to produce biobricks. Current approaches require several pulses of microbes and mineralization media to increase the quantity of calcium carbonate minerals and improve the strength of the material, thus leading to a reduction in sustainability. One potential technique to improve the efficiency of strength development involves trapping the bacteria on the aggregate surfaces using silane coupling agents such as positively charged 3-aminopropyl-methyl-diethoxysilane (APMDES). This treatment traps bacteria on sand through electrostatic interactions that attract negatively charged walls of bacteria to positively charged amine groups. The APMDES treatment promoted an abundant and immediate association of bacteria with sand, increasing the spatial density of ureolytic microbes on sand and promoting efficient initial calcium carbonate precipitation. Though microbial viability was compromised by treatment, urea hydrolysis was minimally affected. Strength was gained much more rapidly for the APMDES-treated sand than for the untreated sand. Three injections of bacteria and biomineralization media using APMDES-treated sand led to the same strength gain as seven injections using untreated sand. The higher strength with APMDES treatment was not explained by increased calcium carbonate accrual in the structure and may be influenced by additional factors such as differences in the microstructure of calcium carbonate bridges between sand particles. Overall, incorporating pretreatment methods, such as amine silane coupling agents, opens a new avenue in biomineralization research by producing materials with an improved efficiency and sustainability.

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).

Experimental studies suggest differences in the distribution of thorax elasticity between insects with synchronous and asynchronous musculature.

Insects have developed diverse flight actuation mechanisms, including synchronous and asynchronous musculature. Indirect actuation, used by insects with both synchronous and asynchronous musculature, transforms thorax exoskeletal deformation into wing rotation. Though thorax deformation is often attributed exclusively to muscle tension, the inertial and aerodynamic forces generated by the flapping wings may also contribute. In this study, a tethered flight experiment was used to simultaneously measure thorax deformation and the inertial/aerodynamic forces acting on the thorax generated by the flapping wing. Compared to insects with synchronous musculature, insects with asynchronous muscle deformed their thorax 60% less relative to their thorax diameter and their wings generated 2.8 times greater forces relative to their body weight. In a second experiment, dorsalventral thorax stiffness was measured across species. Accounting for weight and size, the asynchronous thorax was on average 3.8 times stiffer than the synchronous thorax in the dorsalventral direction. Differences in thorax stiffness and forces acting at the wing hinge led us to hypothesize about differing roles of series and parallel elasticity in the thoraxes of insects with synchronous and asynchronous musculature. Specifically, wing hinge elasticity may contribute more to wing motion in insects with asynchronous musculature than in those with synchronous musculature.

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.

The impacts of biomineralization and oil contamination on the compressive strength of waste plastic-filled mortar.

Researchers have made headway against challenges of increasing cement infrastructure and low plastic recycling rates by using waste plastic in cementitious materials. Past studies indicate that microbially induced calcium carbonate precipitation (MICP) to coat plastic in calcium carbonate may improve the strength. The objective of this study was to increase the amount of clean and contaminated waste plastic that can be added to mortar and to assess whether MICP treatment enhances the strength. The performance of plastic-filled mortar was investigated at 5%, 10%, and 20% volume replacement for cement. Untreated, clean plastics at a 20% cement replacement produced compressive strengths acceptable for several applications. However, a coating of MICP on clean waste plastic did not improve the strengths. At 10% replacement, both MICP treatment and washing of contaminated plastics recovered compressive strengths by approximately 28%, relative to mortar containing oil-coated plastics. By incorporating greater volumes of waste plastics into mortar, the sustainability of cementitious composites has the potential of being improved by the dual mechanisms of reduced cement production and repurposing plastic waste.

Carpenter bee thorax vibration and force generation inform pollen release mechanisms during floral buzzing.

Approximately 10% of flowering plant species conceal their pollen within tube-like poricidal anthers. Bees extract pollen from poricidal anthers via floral buzzing, a behavior during which they apply cyclic forces by biting the anther and rapidly contracting their flight muscles. The success of pollen extraction during floral buzzing relies on the direction and magnitude of the forces applied by the bees, yet these forces and forcing directions have not been previously quantified. In this work, we developed an experiment to simultaneously measure the directional forces and thorax kinematics produced by carpenter bees (Xylocopa californica) during defensive buzzing, a behavior regulated by similar physiological mechanisms as floral buzzing. We found that the buzzing frequencies averaged about 130 Hz and were highly variable within individuals. Force amplitudes were on average 170 mN, but at times reached nearly 500 mN. These forces were 30-80 times greater than the weight of the bees tested. The two largest forces occurred within a plane formed by the bees' flight muscles. Force amplitudes were moderately correlated with thorax displacement, velocity and acceleration amplitudes but only weakly correlated with buzzing frequency. Linear models developed through this work provide a mechanism to estimate forces produced during non-flight behaviors based on thorax kinematic measurements in carpenter bees. Based on the buzzing frequencies, individual bee's capacity to vary buzz frequency and predominant forcing directions, we hypothesize that carpenter bees leverage vibration amplification to increase the deformation of poricidal anthers, and hence the amount of pollen ejected.

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.

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 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.

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.

Biomineralization of Plastic Waste to Improve the Strength of Plastic-Reinforced Cement Mortar.

The development of methods to reuse large volumes of plastic waste is essential to curb the environmental impact of plastic pollution. Plastic-reinforced cementitious materials (PRCs), such as plastic-reinforced mortar (PRM), may be potential avenues to productively use large quantities of low-value plastic waste. However, poor bonding between the plastic and cement matrix reduces the strength of PRCs, limiting its viable applications. In this study, calcium carbonate biomineralization techniques were applied to coat plastic waste and improved the compressive strength of PRM. Two biomineralization treatments were examined: enzymatically induced calcium carbonate precipitation (EICP) and microbially induced calcium carbonate precipitation (MICP). MICP treatment of polyethylene terephthalate (PET) resulted in PRMs with compressive strengths similar to that of plastic-free mortar and higher than the compressive strengths of PRMs with untreated or EICP-treated PET. Based on the results of this study, MICP was used to treat hard-to-recycle types 3-7 plastic waste. No plastics investigated in this study inhibited the MICP process. PRM samples with 5% MICP-treated polyvinyl chloride (PVC) and mixed type 3-7 plastic had compressive strengths similar to plastic-free mortar. These results indicate that MICP treatment can improve PRM strength and that MICP-treated PRM shows promise as a method to reuse plastic waste.

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.

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.

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.

Rational Control of Calcium Carbonate Precipitation by Engineered Escherichia coli.

Ureolytic bacteria ( e.g., Sporosarcina pasteurii) can produce calcium carbonate (CaCO3). Tailoring the size and shape of biogenic CaCO3 may increase the range of useful applications for these crystals. However, wild type Sporosarcina pasteurii is difficult to genetically engineer, limiting control of the organism and its crystal precipitates. Therefore, we designed, constructed, and compared different urease operons and expression levels for CaCO3 production in engineered Escherichia coli strains. We quantified urease expression and calcium uptake and characterized CaCO3 crystal phase and morphology for 13 engineered strains. There was a weak relationship between urease expression and crystal size, suggesting that genes surrounding the urease gene cluster affect crystal size. However, when evaluating strains with a wider range of urease expression levels, there was a negative relationship between urease activity and polycrystal size (e.g., larger crystals with lower activity). The resulting range of crystal morphologies created by the rationally designed strains demonstrates the potential for controlling biogenic CaCO3 precipitation.