Detection of early osteogenic commitment in primary cells using Raman spectroscopy

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm−1) and B-type carbonate (1072 cm−1), DNA (782 cm−1) and collagen matrix (CH2 deformation at 1450 cm−1) and weaker phosphate bands (948 and 970 cm−1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945–952 cm−1) and carbonated apatite (957–962 cm−1) after only 3 days in culture and octacalcium phosphate (970 cm−1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells

Prolonging disuse in aged mice amplifies cortical but not trabecular bones’ response to mechanical loading

Objective: Short-term neurectomy-induced disuse (SN) has been shown to restore load responses in aged mice. We examined whether this restoration was further enhanced in both cortical and trabecular bone by simply extending the SN. Methods: Following load: strain calibration, tibiae in female C57BL/J6 mice at 8, 14 and 20 weeks and 18 months (n=8/group) were loaded and bone changes measured. Effects of long-term SN examined in twenty-six 18 months-old mice, neurectomised for 5 or 100 days with/without subsequent loading. Cortical and trabecular responses were measured histomorphometrically or by micro-computed tomography. Results: Loading increased new cortical bone formation, elevating cross-sectional area in 8, 14 and 20 week-old (p <0.05), but not 18 month-old aged mice. Histomorphometry showed that short-term SN reinstated load-responses in aged mice, with significant 33% and 117% increases in bone accrual at 47% and 37%, but not 27% of tibia length. Cortical responses to loading was heightened and widespread, now evident at all locations, following prolonged SN (108, 167 and 98% at 47, 37 and 27% of tibial length, respectively). In contrast, loading failed to modify trabecular bone mass or architecture. Conclusions: Mechanoadaptation become deficient with ageing and prolonging disuse amplifies this response in cortical but not trabecular bone

Privacy Concerns in Sharing Personal Consumption Data through Online Applications

As online eco-feedback social applications are being increasingly used around the world for motivating citizens to become more energy-aware, privacy concerns in terms of sharing personal electricity consumption data among online contacts are rising. Through Social Electricity, an online application offering personal energy management through a social and collaborative environment, we study the privacy concerns of the users in two case studies, in Cyprus and Singapore. For the Cyprus case, we present an analysis of results gathered through a large questionnaire-based survey combined with mini focus group studies. For the Singapore case, we analyze the findings collected through a students’ semester exercise. This paper provides interesting insights about the overall acceptance and tolerance levels of users of eco-feedback social applications

Vitamin K catabolite inhibition of ovariectomy-induced bone loss: Structure–activity relationship considerations

The potential benefit of vitamin K as a therapeutic in osteoporosis is controversial and the vitamin K regimen being used clinically (45 mg/day) employs doses that are many times higher than required to ensure maximal gamma‐carboxylation of the vitamin K‐dependent bone proteins. We therefore tested the hypothesis that vitamin K catabolites, 5‐carbon (CAN5C) and 7‐carbon carboxylic acid (CAN7C) aliphatic side‐chain derivatives of the naphthoquinone moiety exert an osteotrophic role consistent with the treatment of osteoporosis

Predicting cortical bone adaptation to axial loading in the mouse tibia

The development of predictive mathematical models can contribute to a deeper understanding of the specific stages of bone mechanobiology and the process by which bone adapts to mechanical forces. The objective of this work was to predict, with spatial accuracy, cortical bone adaptation to mechanical load, in order to better understand the mechanical cues that might be driving adaptation. The axial tibial loading model was used to trigger cortical bone adaptation in C57BL/6 mice and provide relevant biological and biomechanical information. A method for mapping cortical thickness in the mouse tibia diaphysis was developed, allowing for a thorough spatial description of where bone adaptation occurs. Poroelastic finite-element (FE) models were used to determine the structural response of the tibia upon axial loading and interstitial fluid velocity as the mechanical stimulus. FE models were coupled with mechanobiological governing equations, which accounted for non-static loads and assumed that bone responds instantly to local mechanical cues in an on–off manner. The presented formulation was able to simulate the areas of adaptation and accurately reproduce the distributions of cortical thickening observed in the experimental data with a statistically significant positive correlation (Kendall's τ rank coefficient τ = 0.51, p < 0.001). This work demonstrates that computational models can spatially predict cortical bone mechanoadaptation to a time variant stimulus. Such models could be used in the design of more efficient loading protocols and drug therapies that target the relevant physiological mechanisms

Modifications of Gait as Predictors of Natural Osteoarthritis Progression in STR/Ort Mice

OBJECTIVE: Osteoarthritis (OA) is a common chronic disease for which disease-modifying therapies are not currently available. Studies to seek new targets for slowing the progress of OA rely on mouse models, but these do not allow for longitudinal monitoring of disease development. This study was undertaken to determine whether gait can be used to measure disease severity in the STR/Ort mouse model of spontaneous OA and whether gait changes are related to OA joint pain. METHODS: Gait was monitored using a treadmill-based video system. Correlations between OA severity and gait at 3 treadmill speeds were assessed in STR/Ort mice. Gait and pain behaviors of STR/Ort mice and control CBA mice were analyzed longitudinally, with monthly assessments. RESULTS: The best speed to identify paw area changes associated with OA severity in STR/Ort mice was found to be 17 cm · seconds(−1). Paw area was modified with age in CBA and STR/Ort mice, but this began earlier in STR/Ort mice and correlated with the onset of OA at 20 weeks of age. In addition, task noncompliance appeared at 20 weeks. Surprisingly, STR/Ort mice did not show any signs of pain with OA development, even when treated with the opioid antagonist naloxone, but did exhibit normal pain behaviors in response to complete Freund's adjuvant–induced arthritis. CONCLUSION: The present results identify an animal model in which OA severity and OA pain can be studied in isolation from one another. The findings suggest that paw area and treadmill noncompliance may be useful tools to longitudinally monitor nonpainful OA development in STR/Ort mice. This will help in providing a noninvasive means of assessing new therapies to slow the progression of OA

A new straightforward method for semi-automated segmentation of trabecular bone from cortical bone in diverse and challenging morphologies

Many physiological, biomechanical, evolutionary and clinical studies that explore skeletal structure and function require successful separation of trabecular from cortical compartments of a bone that has been imaged by X-ray micro-computed tomography (micro-CT) prior to analysis. Separation often involves manual subdivision of these two similarly radio-opaque compartments, which can be time-consuming and subjective. We have developed an objective, semi-automated protocol which reduces user bias and enables straightforward, user-friendly segmentation of trabecular from the cortical bone without requiring sophisticated programming expertise. This method can conveniently be used as a ‘recipe’ in commercial programmes (Avizo herein) and applied to a variety of datasets. Here, we characterize and share this recipe, and demonstrate its application to a range of murine and human bone types, including normal and osteoarthritic specimens, and bones with distinct embryonic origins and spanning a range of ages. We validate the method by testing inter-user bias during the scan preparation steps and confirm utility in the architecturally challenging analysis of growing murine epiphyses. We also report details of the recipe, so that other groups can readily re-create a similar method in open access programmes. Our aim is that this method will be adopted widely to create a reproducible and time-efficient method of segmenting trabecular and cortical bone

Lasting organ-level bone mechanoadaptation is unrelated to local strain

Bones adapt to mechanical forces according to strict principles predicting straight shape. Most bones are, however, paradoxically curved. To solve this paradox, we used computed tomography–based, four-dimensional imaging methods and computational analysis to monitor acute and chronic whole-bone shape adaptation and remodeling in vivo. We first confirmed that some acute load-induced structural changes are reversible, adhere to the linear strain magnitude regulation of remodeling activities, and are restricted to bone regions in which marked antiresorptive actions are evident. We make the novel observation that loading exerts significant lasting modifications in tibial shape and mass across extensive bone regions, underpinned by (re)modeling independent of local strain magnitude, occurring at sites where the initial response to load is principally osteogenic. This is the first report to demonstrate that bone loading stimulates nonlinear remodeling responses to strain that culminate in greater curvature adjusted for load predictability without sacrificing strength