Spatial relationship between bone formation and mechanical stimulus within cortical bone: Combining 3D fluorochrome mapping and poroelastic finite element modelling.

Bone is a dynamic tissue and adapts its architecture in response to biological and mechanical factors. Here we investigate how cortical bone formation is spatially controlled by the local mechanical environment in the murine tibia axial loading model (C57BL/6). We obtained 3D locations of new bone formation by performing 'slice and view' 3D fluorochrome mapping of the entire bone and compared these sites with the regions of high fluid velocity or strain energy density estimated using a finite element model, validated with ex-vivo bone surface strain map acquired ex-vivo using digital image correlation. For the comparison, 2D maps of the average bone formation and peak mechanical stimulus on the tibial endosteal and periosteal surface across the entire cortical surface were created. Results showed that bone formed on the periosteal and endosteal surface in regions of high fluid flow. Peak strain energy density predicted only the formation of bone periosteally. Understanding how the mechanical stimuli spatially relates with regions of cortical bone formation in response to loading will eventually guide loading regime therapies to maintain or restore bone mass in specific sites in skeletal pathologies.

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide-mediated vasorelaxation in BALB/c mice.

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L-NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1-34] (80 µg/kg/day) or L-NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro-CT, histomorphometry and three-point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co-treatment with L-NAME blocked the action of PTH on blood flow, whereas L-NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co-treatment with L-NAME restricted the PTH-stimulated increase in cortical bone formation but had no clear-cut effects in trabecular bone. Co-treatment with L-NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO-mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone.

Excessive growth hormone expression in male GH transgenic mice adversely alters bone architecture and mechanical strength.

Patients with acromegaly have a higher prevalence of vertebral fractures despite normal bone mineral density (BMD), suggesting that GH overexpression has adverse effects on skeletal architecture and strength. We used giant bovine GH (bGH) transgenic mice to analyze the effects of high serum GH levels on BMD, architecture, and mechanical strength. Five-month-old hemizygous male bGH mice were compared with age- and sex-matched nontransgenic littermates controls (NT; n=16/group). Bone architecture and BMD were analyzed in tibia and lumbar vertebrae using microcomputed tomography. Femora were tested to failure using three-point bending and bone cellular activity determined by bone histomorphometry. bGH transgenic mice displayed significant increases in body weight and bone lengths. bGH tibia showed decreases in trabecular bone volume fraction, thickness, and number compared with NT ones, whereas trabecular pattern factor and structure model index were significantly increased, indicating deterioration in bone structure. Although cortical tissue perimeter was increased in transgenic mice, cortical thickness was reduced. bGH mice showed similar trabecular BMD but reduced trabecular thickness in lumbar vertebra relative to controls. Cortical BMD and thickness were significantly reduced in bGH lumbar vertebra. Mechanical testing of femora confirmed that bGH femora have decreased intrinsic mechanical properties compared with NT ones. Bone turnover is increased in favor of bone resorption in bGH tibia and vertebra compared with controls, and serum PTH levels is also enhanced in bGH mice. These data collectively suggest that high serum GH levels negatively affect bone architecture and quality at multiple skeletal sites.

Refine, reduce, replace: Imaging of fibrosis and arthritis in animal models.

Non-invasive imaging has great potential to contribute to the 'Three R's' principles for more ethical use of experimental animals. It enables repetitive monitoring of disease progression and measurement of quantitative biomarkers that report on disease progression and therapy efficacy in the same animal, thereby reducing manifold the number of animals needed for in vivo studies whilst advancing our knowledge into the pathophysiology of these diseases. This article reviews applications of non-invasive imaging in the field of fibrosis and arthritis research. It provides evidence for the viability of this approach not only for ethical reasons (reducing numbers and suffering in research animals, according to the 3R principles) but also for accelerating experimental output and making it more translational. The emphasis is on promising developments which will help improving throughput by reducing experiment length and size as well as human resources for data analysis, therefore encouraging a wider spreading of novel imaging technologies.

Utility of a Liquid Formulation of Levo-thyroxine in Differentiated Thyroid Cancer Patients.

The goal of levo-thyroxine (L-T4) administration in differentiated thyroid cancer (DTC) is to suppress thyroid stimulating hormone (TSH) levels. The tolerability and efficacy of a new formulation of liquid L-T4 vs. the previous tablet formulation was evaluated in a cohort of 59 patients with cured DTC. The correlation between breakfast modality and therapy was also monitored. Hormonal and clinical evaluations were performed before and 70 days after patients were switched from tablet to liquid L-T4 formulation, without changes in daily dose. Breakfast habits were evaluated. The interval between L-T4 therapy and breakfast was recorded. Patient approval of L-T4 formulations was evaluated. 8% of patients dropped out owing to adverse events. The modality of L-T4 administration proved adequate under tablet and liquid formulation in 64% and 68% of patients who fully complied with the protocol. While significantly more patients found the tablet formulation more agreeable, at the end of the protocol subjective symptoms had diminished significantly and 73% requested to remain on the liquid formulation. No change in TSH, thyroid hormones or thyroglobulin was noted during the study. A balanced breakfast containing less than 4 g of alimentary fibre did not interfere with L-T4 therapy. Liquid L-T4 seems to be a valid alternative formulation in DTC patients, its initial dislike being outweighed by a significant reduction in subjective symptoms. Both tablet and liquid L-T4 therapy require monitoring over time. A continental breakfast containing less than 4 g of alimentary fibres seems to favour the absorption of L-T4, whether in tablet or liquid formulation.

Rapid, automated imaging of mouse articular cartilage by microCT for early detection of osteoarthritis and finite element modelling of joint mechanics.

OBJECTIVE: Mouse articular cartilage (AC) is mostly assessed by histopathology and its mechanics is poorly characterised. In this study: (1) we developed non-destructive imaging for quantitative assessment of AC morphology and (2) evaluated the mechanical implications of AC structural changes. METHODS: Knee joints obtained from naïve mice and from mice with osteoarthritis (OA) induced by destabilization of medial meniscus (DMM) for 4 and 12 weeks, were imaged by phosphotungstic acid (PTA) contrast enhanced micro-computed tomography (PTA-CT) and scored by conventional histopathology. Our software (Matlab) automatically segmented tibial AC, drew two regions centred on each tibial condyle and evaluated the volumes included. A finite element (FE) model of the whole mouse joint was implemented to evaluate AC mechanics. RESULTS: Our method achieved rapid, automated analysis of mouse AC (structural parameters in <10 h from knee dissection) and was able to localise AC loss in the central region of the medial tibial condyle. AC thickness decreased by 15% at 4 weeks and 25% at 12 weeks post DMM surgery, whereas histopathology scores were significantly increased only at 12 weeks. FE simulations estimated that AC thinning at early-stages in the DMM model (4 weeks) increases contact pressures (+39%) and Tresca stresses (+43%) in AC. CONCLUSION: PTA-CT imaging is a fast and simple method to assess OA in murine models. Once applied more extensively to confirm its robustness, our approach will be useful for rapidly phenotyping genetically modified mice used for OA research and to improve the current understanding of mouse cartilage mechanics.

Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.

OBJECTIVE: Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX). METHODS: Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed. RESULTS: In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume +117% versus Ctrl-Ab-treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (+24% and +20%, respectively, versus Ctrl-Ab-treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEX-treated mice (maximum load +60% and ultimate strength +47% in Scl-AbI-treated mice versus Ctrl-Ab-treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab. CONCLUSION: Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice.

Sympathetic nervous system and bone adaptive response to its mechanical environment.

While bone adaptive response to its mechanical environment was considered to be controlled locally by cytokines and systemic hormones, some recent work suggests that it could also be neuronally regulated. Bone is indeed very densely innervated and many experimental and clinical studies have previously shown the involvement of the nervous system in the control of bone metabolism. The demonstration that the central nervous system regulates bone mass via the sympathetic nervous system (SNS) has prompted recent studies aimed to investigate the role of the SNS in the bone mechano-adaptive response. This review will focus on this work and summarize the evidence for a contribution of the beta-adrenergic signalling in the response of bone cells to mechanical loading. The apparent conflicting results obtained in diverse experimental models of loading and unloading, at different skeletal sites, and in relation to various hormonal levels, will be discussed. While those studies do not support a major influence of the SNS on the bone mechano-adaptive response, there is nevertheless strong evidence that the SNS is part of a complex system which contributes to the metabolic regulation of bone.

A collagen-based interface construct for the assessment of cell-dependent mechanical integration of tissue surfaces.

The interface between any newly engineered tissue and pre-existing tissue is of great importance to tissue engineering; however, this process has so far been largely ignored, with few reports regarding the mechanical strength of newly integrated connective tissues surfaces. A new model system has been developed to generate a well-defined interface between two collagen lattices: one pre-contracted by resident fibroblasts and the other a cell-free wrapping gel. This construct can be cultured for prolonged periods (>2 weeks) and can also be fitted onto a mechanical testing system to measure the interface adhesive strength at the end of the culture time. Interface adhesive strength shows a six-fold increase after 1 week in culture, compared with the time-zero baseline. Observations of cell migration across the interface suggest that cell translocation in the three-dimensional matrix might play an important role in the integration process. In this new controlled geometry, normal and shear stresses at the interface can be analysed by finite element modelling and the areas at which debonding starts can be defined. The current experimental design permits solid multiple (homogeneous or heterogeneous) interface formation in vitro with a well-defined geometry and the possibility of measuring mechanical linkage. This design should enable many other factors affecting cell-driven interface strengthening to be investigated.

Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not due to resorption.

The current study tests the hypothesis that basal level and minute-by-minute correction of plasma Ca2+ by outward and inward Ca2+ fluxes from and into an exchangeable ionic pool in bone is controlled by an active partition system without contributions from the bone remodeling system. Direct real-time measurements of Ca2+ fluxes were made using the scanning ion-selective electrode technique (SIET) on living bones maintained ex vivo in physiological conditions. SIET three-dimensional measurements of the local Ca2+ concentration gradient (10 microm spatial resolution) were performed on metatarsal bones of weanling mice after drilling a 100-mum hole through the cortex to expose the internal bone extracellular fluid (BECF) to the bathing solution, whose composition mimicked the extracellular fluid (ECF). Influxes of Ca2+ towards the center of the cortical hole (15.1+/-4.2 pmol cm-2 s-1) were found in the ECF and were reversed to effluxes (7.4+/-2.9 pmol cm-2 s-1) when calcium was depleted from the ECF, mimicking a plasma demand. The reversal from influx to efflux and vice versa was immediate and fluxes in both directions were steady throughout the experimental time (>or=2 h, n=14). Only the efflux was nullified within 10 min by the addition of 10 mM/L Na-Cyanide (n=7), demonstrating its cell dependence. The timeframes of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable calcium pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play a central role in the short-term error correction of plasma calcium without the contribution of bone remodeling.

Optical measurement of three-dimensional collagen gel constructs by elastic scattering spectroscopy.

Analysis of the formation and organization of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology. We have analyzed early-stage responses in collagen lattices, using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions. The method is based on an optical system in which an optical fiber delivers white light onto the tissue and the back-scattered light is collected for spectroscopy by another optical fiber. Results show correlation between changes in the spectral signatures with changes in the collagen gel contraction or internal organization in all three models of collagen construct analyzed. Therefore elastic scattering spectroscopy is a promising tool to monitor tissue-engineered constructs or early repair in collagenous tissues.