Inactivation of Salmonella in nonintact beef during low-temperature sous vide cooking.

Sous vide cooking is a method of food preparation in which food is vacuum sealed and cooked in a water bath that is set to a precise temperature and circulated by a sous vide device. Due to ease of use and affordability, this cooking method has grown increasingly popular in food service kitchens and domestic settings. However, low-temperature, long holding time sous vide cooking recommendations from manufacturers and chefs in popular press raise food safety concerns - specifically those for the preparation of nonintact beef products. The objective of this experiment was to address these concerns by validating a 5 log reduction of Salmonella spp. in sous vide cooked, nonintact beef steaks. Beef semitendinosus sliced into 2.54 cm steaks were internally inoculated to 7 log with Salmonella Typhimurium, Enteritidis, and Heidelberg via a needle inoculation pin pad. Steaks were individually vacuum sealed, and sous vide cooked at 46.1, 51.6, and 54.4°C. The minimum time measured for a 5 log reduction at 51.6 and 54.4°C was 150 and 64.5 min, respectively (P < 0.01). Additionally, a 7.28 log final reduction was achieved at 51.6°C after 322.5 min (P < 0.01). However, 46.1°C was only able to achieve a final reduction of 2.01 log (P < 0.01) after a holding time of 420 min. The results of this experiment validate in sous vide cooked products the time and temperature combinations provided in the USDA-FSIS Appendix A guidance for a 5 log reduction of Salmonella spp. in meat products. Moreover, more research is needed with other relevant foodborne pathogens to determine if sous vide cooking below Appendix A recommendations could lead to unsafe products.

Distributions of Microdamage Are Altered Between Trabecular Rods and Plates in Cancellous Bone From Men With Type 2 Diabetes Mellitus.

Individuals with type 2 diabetes mellitus (T2DM) have an increased risk of fragility fracture despite exhibiting normal to high bone mineral density (BMD). Conditions arising from T2DM, such as reduced bone turnover and alterations in microarchitecture, may contribute to skeletal fragility by influencing bone morphology and microdamage accumulation. The objectives of this study were (i) to characterize the effect of T2DM on microdamage quantity and morphology in cancellous bone, and (ii) relate the accumulation of microdamage to the cancellous microarchitecture. Cancellous specimens from the femoral neck were collected during total hip arthroplasty (T2DM: n = 22, age = 65 ± 9 years, glycated hemoglobin [HbA1c] = 7.00% ± 0.98%; non-diabetic [non-DM]: n = 25, age = 61 ± 8 years, HbA1c = 5.50% ± 0.4%), compressed to 3% strain, stained with lead uranyl acetate to isolate microdamage, and scanned with micro-computed tomography (µCT). Individual trabeculae segmentation was used to isolate rod-like and plate-like trabeculae and their orientations with respect to the loading axis. The T2DM group trended toward a greater BV/TV (+27%, p = 0.07) and had a more plate-like trabecular architecture (+8% BVplates , p = 0.046) versus non-DM specimens. Rods were more damaged relative to their volume compared to plates in the non-DM group (DVrods /BVrods versus DVplates /BVplates : +49%, p < 0.0001), but this difference was absent in T2DM specimens. Longitudinal rods were more damaged in the non-DM group (DVlongitudinal rods /BVlongitudinal rods : +73% non-DM versus T2DM, p = 0.027). Total damage accumulation (DV/BV) and morphology (DS/DV) did not differ in T2DM versus non-DM specimens. These results provide evidence that cancellous microarchitecture does not explain fracture risk in T2DM, pointing to alterations in material matrix properties. In particular, cancellous bone from men with T2DM may have an attenuated ability to mitigate microdamage accumulation through sacrificial rods. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone Tissue Composition in Postmenopausal Women Varies With Glycemic Control From Normal Glucose Tolerance to Type 2 Diabetes Mellitus.

The risk of fragility fracture increases for people with type 2 diabetes mellitus (T2DM), even after controlling for bone mineral density, body mass index, visual impairment, and falls. We hypothesize that progressive glycemic derangement alters microscale bone tissue composition. We used Fourier-transform infrared (FTIR) imaging to analyze the composition of iliac crest biopsies from cohorts of postmenopausal women characterized by oral glucose tolerance testing: normal glucose tolerance (NGT; n = 35, age = 65 ± 7 years, HbA1c = 5.8 ± 0.3%), impaired glucose tolerance (IGT; n = 26, age = 64 ± 5 years, HbA1c = 6.0 ± 0.4%), and overt T2DM on insulin (n = 25, age = 64 ± 6 years, HbA1c = 9.13 ± 0.6). The distributions of cortical bone mineral content had greater mean values (+7%) and were narrower (-10%) in T2DM versus NGT groups (p < 0.05). The distributions of acid phosphate, an indicator of new mineral, were narrower in cortical T2DM versus NGT and IGT groups (-14% and -14%, respectively) and in trabecular NGT and IGT versus T2DM groups (-11% and -10%, respectively) (all p < 0.05). The distributions of crystallinity were wider in cortical NGT versus T2DM groups (+16%) and in trabecular NGT versus T2DM groups (+14%) (all p < 0.05). Additionally, bone turnover was lower in T2DM versus NGT groups (P1NP: -25%, CTx: -30%, ucOC: -24%). Serum pentosidine was similar across groups. The FTIR compositional and biochemical marker values of the IGT group typically fell between the NGT and T2DM group values, although the differences were not always statistically significant. In summary, worsening glycemic control was associated with greater mineral content and narrower distributions of acid phosphate, an indicator of new mineral, which together are consistent with observations of lower turnover; however, wider distributions of mineral crystallinity were also observed. A more mineralized, less heterogeneous tissue may affect tissue-level mechanical properties and in turn degrade macroscale skeletal integrity. In conclusion, these data are the first evidence of progressive alteration of bone tissue composition with worsening glycemic control in humans. © 2020 American Society for Bone and Mineral Research (ASBMR).

Effects of Diabetes on Bone Material Properties.

PURPOSE OF REVIEW: Individuals with type 1 and type 2 diabetes mellitus (T1DM, T2DM) have an increased risk of bone fracture compared to non-diabetic controls that is not explained by differences in BMD, BMI, or falls. Thus, bone tissue fracture resistance may be reduced in individuals with DM. The purpose of this review is to summarize work that analyzes the effects of T1DM and T2DM on bone tissue compositional and mechanical properties. RECENT FINDINGS: Studies of clinical T2DM specimens revealed increased mineralization and advanced glycation endproduct (AGE) concentrations and significant relationships between mechanical performance and composition of cancellous bone. Specifically, in femoral cancellous tissue, compressive stiffness and strength increased with mineral content; and post-yield properties decreased with AGE concentration. In addition, cortical resistance to in vivo indentation (bone material strength index) was lower in patients with T2DM vs. age-matched non-diabetic controls, and this resistance decreased with worsening glycemic control. Recent studies on patients with T1DM and history of a prior fragility fracture found greater mineral content and concentrations of AGEs in iliac trabecular bone and correspondingly stiffer, harder bone at the nanosacle. Recent observational data showed greater AGE and mineral content in surgically retrieved bone from patients with T2DM vs. non-DM controls, consistent with reduced bone remodeling. Limited data on human T1DM bone tissue also showed higher mineral and AGE content in patients with prior fragility fractures compared to non-DM and non-fracture controls.

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.

Altered Tissue Composition, Microarchitecture, and Mechanical Performance in Cancellous Bone From Men With Type 2 Diabetes Mellitus.

People with type 2 diabetes mellitus (T2DM) have normal-to-high BMDs, but, counterintuitively, have greater fracture risks than people without T2DM, even after accounting for potential confounders like BMI and falls. Therefore, T2DM may alter aspects of bone quality, including material properties or microarchitecture, that increase fragility independently of bone mass. Our objective was to elucidate the factors that influence fragility in T2DM by comparing the material properties, microarchitecture, and mechanical performance of cancellous bone in a clinical population of men with and without T2DM. Cancellous specimens from the femoral neck were collected during total hip arthroplasty (T2DM: n = 31, age = 65 ± 8 years, HbA1c = 7.1 ± 0.9%; non-DM: n = 34, age = 62 ± 9 years, HbA1c = 5.5 ± 0.4%). The T2DM specimens had greater concentrations of the advanced glycation endproduct pentosidine (+ 36%, P < 0.05) and sugars bound to the collagen matrix (+ 42%, P < 0.05) than the non-DM specimens. The T2DM specimens trended toward a greater bone volume fraction (BV/TV) (+ 24%, NS, P = 0.13) and had greater mineral content (+ 7%, P < 0.05) than the non-DM specimens. Regression modeling of the mechanical outcomes revealed competing effects of T2DM on bone mechanical behavior. The trend of higher BV/TV values and the greater mineral content observed in the T2DM specimens increased strength, whereas the greater values of pentosidine in the T2DM group decreased postyield strain and toughness. The long-term medical management and presence of osteoarthritis in these patients may influence these outcomes. Nevertheless, our data indicate a beneficial effect of T2DM on cancellous microarchitecture, but a deleterious effect of T2DM on the collagen matrix. These data suggest that high concentrations of advanced glycation endproducts can increase fragility by reducing the ability of bone to absorb energy before failure, especially for the subset of T2DM patients with low BV/TV. © 2019 American Society for Bone and Mineral Research.

Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves.

Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. STATEMENT OF SIGNIFICANCE: We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

Bone Tissue Collagen Maturity and Mineral Content Increase With Sustained Hyperglycemia in the KK-Ay Murine Model of Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) increases fracture risk for a given bone mineral density (BMD), which suggests that T2DM changes bone tissue properties independently of bone mass. In this study, we assessed the effects of hyperglycemia on bone tissue compositional properties, enzymatic collagen crosslinks, and advanced glycation end-products (AGEs) in the KK-Ay murine model of T2DM using Fourier transform infrared (FTIR) imaging and high-performance liquid chromatography (HPLC). Compared to KK-aa littermate controls (n = 8), proximal femoral bone tissue of KK-Ay mice (n = 14) exhibited increased collagen maturity, increased mineral content, and less heterogeneous mineral properties. AGE accumulation assessed by the concentration of pentosidine, as well as the concentrations of the nonenzymatic crosslinks hydroxylysylpyridinoline (HP) and lysyl pyridinoline (LP), did not differ in the proximal femurs of KK-Ay mice compared to controls. The observed differences in tissue-level compositional properties in the KK-Ay mice are consistent with bone that is older and echo observations of reduced remodeling in T2DM. © 2017 American Society for Bone and Mineral Research.

Bone quality assessment techniques: geometric, compositional, and mechanical characterization from macroscale to nanoscale.

This review presents an overview of the characterization techniques available to experimentally evaluate bone quality, defined as the geometric and material factors that contribute to fracture resistance independently of areal bone mineral density (aBMD) assessed by dual energy x-ray absorptiometry. The methods available for characterization of the geometric, compositional, and mechanical properties of bone across multiple length scales are summarized, along with their outcomes and their advantages and disadvantages. Examples of how each technique is used are discussed, as well as practical concerns such as sample preparation and whether or not each testing method is destructive. Techniques that can be used in vivo and those that have been recently improved or developed are emphasized, including high resolution peripheral quantitative computed tomography to evaluate geometric properties and reference point indentation to evaluate material properties. Because no single method can completely characterize bone quality, we provide a framework for how multiple characterization methods can be used together to generate a more comprehensive analysis of bone quality to complement aBMD in fracture risk assessment.