Antonia Torcasio

Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor-α

Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor‐alpha (ERα) following aromatization of androgens into estrogens. To assess the differential action of AR and ERα pathways on bone and body composition, AR‐ERα double‐knockout mice were gener‐ated and characterized. AR disruption decreased trabec‐ular bone mass, whereas ERα disruption had no additional effect on the AR‐dependent trabecular bone loss. In contrast, combined AR and ERα inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERα disruption alone. ERα inactivation—in the presence or absence of AR—increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERα activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERα activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERα signaling on trabecu‐lar and cortical bone mass as well as body composition in male mice.—Callewaert, F., Venken, K., Ophoff, J., De Gendt, K., Torcasio, A., van Lenthe, G. H., Van Ooster‐wyck, H., Boonen, S., Bouillon, R., Verhoeven, G., Vanderschueren, D. Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor‐α. FASEB J. 23, 232‐240 (2009)

Sexual Dimorphism in Cortical Bone Size and Strength But Not Density Is Determined by Independent and Time-Specific Actions of Sex Steroids and IGF-1: Evidence From Pubertal Mouse Models

Although it is well established that males acquire more bone mass than females, the underlying mechanism and timing of this sex difference remain controversial. The aim of this study was to assess the relative contribution of sex steroid versus growth hormone–insulin‐like growth factor 1 (GH–IGF‐1) action to pubertal bone mass acquisition longitudinally in pubertal mice. Radial bone expansion peaked during early puberty (3 to 5 weeks of age) in male and female mice, with significantly more expansion in males than in females (+40%). Concomitantly, in 5 week old male versus female mice, periosteal and endocortical bone formation was higher (+70%) and lower (−47%), respectively, along with higher serum IGF‐1 levels during early puberty in male mice. In female mice, ovariectomy increased radial bone expansion during early puberty as well as the endocortical perimeter. In male mice, orchidectomy reduced radial bone expansion only during late puberty (5 to 8 weeks of age), whereas combined androgen and estrogen deficiency modestly decreased radial bone expansion during early puberty, accompanied by lower IGF‐1 levels. GHRKO mice with very low IGF‐1 levels, on the other hand, showed limited radial bone expansion and no skeletal dimorphism. From these data we conclude that skeletal sexual dimorphism is established during early puberty and depends primarily on GH–IGF‐1 action. In males, androgens and estrogens have stimulatory effects on bone size during late and early puberty, respectively. In females, estrogens limit bone size during early puberty. These longitudinal findings in mice provide strong evidence that skeletal dimorphism is determined by independent and time‐specific effects of sex steroids and IGF‐1.

In vivo assessment of the effect of controlled high- and low-frequency mechanical loading on peri-implant bone healing

The aim of this study was to investigate the effect of controlled high- (HF) and low-frequency (LF) mechanical loading on peri-implant bone healing. Custom-made titanium implants were inserted in both tibiae of 69 adult Wistar rats. For every animal, one implant was loaded by compression through the axis of tibia (test), whereas the other one was unloaded (control). The test implants were randomly distributed among four groups receiving different loading regimes, which were determined by ex vivo calibration. Within the HF (40 Hz) or LF (2 Hz) loading category, the magnitudes were chosen as low- (LM) and high-magnitude (HM), respectively, leading to constant strain rate amplitudes for the two frequency groups. This resulted in the four loading regimes: (i) HF-LM (40 Hz–0.5 N); (ii) HF-HM (40 Hz–1 N); (iii) LF-LM (2 Hz–10 N); and (iv) LF-HM (2 Hz–20 N) loading. Loading was performed five times per week and lasted for one or four weeks. Tissue samples were processed for histology and histomorphometry (bone-to-implant contact, BIC; and peri-implant bone fraction, BF) at the cortical and medullar level. Data were analysed statistically with ANOVA and paired t-tests with the significance level set at 0.05. For the one-week experiments, an increased BF adjacent to the implant surface at the cortical level was exclusively induced by the LF-HM loading regime (2 Hz–20 N). Four weeks of loading resulted in a significant effect on BIC (and not on BF) in case of HF-LM loading (40 Hz–0.5 N) and LF-HM loading (2 Hz–20 N): BIC at the cortical level significantly increased under both loading regimes, whereas BIC at the medullar level was positively influenced only in case of HF-LM loading. Mechanical loading at both HF and LF affects osseointegration and peri-implant BF. Higher loading magnitudes (and accompanying elevated tissue strains) are required under LF loading to provoke a positive peri-implant bone response, compared with HF loading. A sustained period of loading at HF is needed to result in an overall enhanced osseointegration.

Enhancement of implant osseointegration by high-frequency low-magnitude loading.

Background Mechanical loading is known to play an important role in bone remodelling. This study aimed to evaluate the effect of high- and low-frequency axial loading, applied directly to the implant, on peri-implant bone healing and implant osseointegration. Methodology Titanium implants were bilaterally installed in rat tibiae. For every animal, one implant was loaded (test) while the other one was not (control). The test implants were randomly divided into 8 groups according to 4 loading regimes and 2 experimental periods (1 and 4 weeks). The loaded implants were subject to an axial displacement. Within the high- (HF, 40 Hz) or low-frequency (LF, 8 Hz) loading category, the displacements varied 2-fold and were ranked as low- or high-magnitude (LM, HM), respectively. The strain rate amplitudes were kept constant between the two frequency groups. This resulted in the following 4 loading regimes: 1) HF-LM, 40 Hz-8 µm; 2) HF-HM, 40 Hz-16 µm; 3) LF-LM, 8 Hz-41 µm; 4) LF-HM, 8 Hz-82 µm. The tissue samples were processed for resin embedding and subjected to histological and histomorphometrical analyses. Data were analyzed statistically with the significance set at p<0.05. Principal Findings After loading for 4 weeks, HF-LM loading (40 Hz-8 µm) induced more bone-to-implant contact (BIC) at the level of the cortex compared to its unloaded control. No significant effect of the four loading regimes on the peri-implant bone fraction (BF) was found in the 2 experimental periods. Conclusions The stimulatory effect of immediate implant loading on bone-to-implant contact was only observed in case of high-frequency (40 Hz) low-magnitude (8 µm) loading. The applied load regimes failed to influence the peri-implant bone mass.

Comparison of optical coherence tomography, microcomputed tomography, and histology at a three-dimensionally imaged trabecular bone sample.

We investigate optical coherence tomography (OCT) as a method for imaging bone. The OCT images are compared directly to those of the standard methods of bone histology and microcomputed tomography (microCT) on a single, fixed human femoral trabecular bone sample. An advantage of OCT over bone histology is its noninvasive nature. OCT also images the lamellar structure of trabeculae at slightly higher contrast than normal bone histology. While microCT visualizes the trabecular framework of the whole sample, OCT can image additionally cells with a penetration depth limited approximately to 1 mm. The most significant advantage of OCT, however, is the absence of toxic effects (no ionizing radiation), i.e., continuous images may be made and individual cell tracking may be performed. The penetration depth of OCT, however, limits its use to small animal models and small bone organ cultures.

Trabecular Bone Adaptation to Low-Magnitude High-Frequency Loading in Microgravity

Exposure to microgravity causes loss of lower body bone mass in some astronauts. Low-magnitude high-frequency loading can stimulate bone formation on earth. Here we hypothesized that low-magnitude high-frequency loading will also stimulate bone formation under microgravity conditions. Two groups of six bovine cancellous bone explants were cultured at microgravity on a Russian Foton-M3 spacecraft and were either loaded dynamically using a sinusoidal curve or experienced only a static load. Comparable reference groups were investigated at normal gravity. Bone structure was assessed by histology, and mechanical competence was quantified using μCT and FE modelling; bone remodelling was assessed by fluorescent labelling and secreted bone turnover markers. Statistical analyses on morphometric parameters and apparent stiffness did not reveal significant differences between the treatment groups. The release of bone formation marker from the groups cultured at normal gravity increased significantly from the first to the second week of the experiment by 90.4% and 82.5% in response to static and dynamic loading, respectively. Bone resorption markers decreased significantly for the groups cultured at microgravity by 7.5% and 8.0% in response to static and dynamic loading, respectively. We found low strain magnitudes to drive bone turnover when applied at high frequency, and this to be valid at normal as well as at microgravity. In conclusion, we found the effect of mechanical loading on trabecular bone to be regulated mainly by an increase of bone formation at normal gravity and by a decrease in bone resorption at microgravity. Additional studies with extended experimental time and increased samples number appear necessary for a further understanding of the anabolic potential of dynamic loading on bone quality and mechanical competence.

TRABECULAR BONE ADAPTATION TO LOW-MAGNITUDE HIGH-FREQUENCY LOADING AT MICRO-GRAVITY

Exposure to microgravity causes loss of lower body bone mass in some astronauts. Low-magnitude high-frequency loading can stimulate bone formation on earth. Here we hypothesized that low-magnitude high-frequency loading will also stimulate bone formation under microgravity conditions. Two groups of six bovine cancellous bone explants were cultured at microgravity on a Russian Foton-M3 spacecraft and were either loaded dynamically using a sinusoidal curve or experienced only a static load. Comparable reference groups were investigated at normal gravity. Bone structure was assessed by histology, and mechanical competence was quantified using mCT and FE modelling; bone remodelling was assessed by fluorescent labelling and secreted bone turnover markers. Statistical analyses on morphometric parameters and apparent stiffness did not reveal significant differences between the treatment groups. The release of bone formation marker from the groups cultured at normal gravity increased significantly from the first to the second week of the experiment by 90.4% and 82.5% in response to static and dynamic loading, respectively. Bone resorption markers decreased significantly for the groups cultured at microgravity by 7.5% and 8.0% in response to static and dynamic loading, respectively. We found low strain magnitudes to drive bone turnover when applied at high frequency, and this to be valid at normal as well as at microgravity. In conclusion, we found the effect of mechanical loading on trabecular bone to be regulated mainly by an increase of bone formation at normal gravity and by a decrease in bone resorption at microgravity. Additional studies with extended experimental time and increased samples number appear necessary for a further understanding of the anabolic potential of dynamic loading on bone quality and mechanical competence. Citation: Torcasio A, Jahn K, Van Guyse M, Spaepen P, Tami AE, et al. (2014) Trabecular Bone Adaptation to Low-Magnitude High-Frequency Loading in Microgravity. PLoS ONE 9(5): e93527. doi:10.1371/journal.pone.0093527 Editor: Ali Al-Ahmad, University Hospital of the Albert-Ludwigs-University Freiburg, Germany Received May 22, 2013; Accepted March 7, 2014; Published May 2, 2014 Copyright: 2014 Torcasio et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by grant C90346 (Belgium PRODEX-9 project) and the ESA MAP grant #AO99-122. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: harry.vanlenthe@mech.kuleuven.be

Three-dimensional bone imaging: optical coherence tomography versus micro computer tomography

Optical coherence tomography (OCT) and micro-computed tomography (μCT) were applied to a bone sample, a 3x4x4mm cube of fixed substantia spongiosa from an arthritic human hip. Three-dimensional image sets (1.0mm x 0.9mm x 1.6mm) were acquired with both imaging systems for the same volume of interest. For better navigation, the sample surface was additionally imaged with microscopy. The resulting OCT images were compared stepwise to the according μCT images, showing a high correlation regarding the visualization of individual trabeculae. System based imaging differences were also found: due to scattering, OCT is limited to an imaging depth of about 1mm, while μCT is capable of imaging the complete trabecular bone architecture. However, OCT images cells and the inner bone structures in contrast to μCT at similar nominal resolutions (5μm respectively 6.5μm).