Gaël Y. Rochefort

Osteocyte: the unrecognized side of bone tissue

IntroductionOsteocytes represent 95% of all bone cells. These cells are old osteoblasts that occupy the lacunar space and are surrounded by the bone matrix. They possess cytoplasmic dendrites that form a canalicular network for communication between osteocytes and the bone surface. They express some biomarkers (osteopontin, β3 integrin, CD44, dentin matrix protein 1, sclerostin, phosphate-regulating gene with homologies to endopeptidases on the X chromosome, matrix extracellular phosphoglycoprotein, or E11/gp38) and have a mechano-sensing role that is dependent upon the frequency, intensity, and duration of strain.DiscussionThe mechanical information transmitted into the cytoplasm also triggers a biological cascade, starting with NO and PGE2 and followed by Wnt/β catenin signaling. This information is transmitted to the bone surface through the canalicular network, particularly to the lining cells, and is able to trigger bone remodeling by directing the osteoblast activity and the osteoclastic resorption. Furthermore, the osteocyte death seems to play also an important role. The outcome of micro-cracks in the vicinity of osteocytes may interrupt the canalicular network and trigger cell apoptosis in the immediate surrounding environment. This apoptosis appears to transmit a message to the bone surface and activate remodeling. The osteocyte network also plays a recognized endocrine role, particularly concerning phosphate regulation and vitamin D metabolism. Both the suppression of estrogen following menopause and chronic use of systemic glucocorticoids induce osteocyte apoptosis. On the other hand, physical activity has a positive impact in the reduction of apoptosis. In addition, some osteocyte molecular elements like sclerostin, connexin 43, E11/gp38, and DKK1 are emerging as promising targets for the treatment of various osteo-articular pathologies.

Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia.

INTRODUCTION Alcohol is known to decrease bone mineral density (BMD) and to induce trabecular microarchitecture deterioration. However, little is known about the effects of chronic alcohol consumption on osteocytes in situ. The aim of this study was to assess the effects of a high alcohol dose on osteocytes in an alcohol-induced osteopenia model. MATERIALS AND METHODS 24 male Wistar rats, 2-months old were separated in 2 groups: Control (C) or Alcohol (A35). The rats in the A35 group drank a beverage composed of 35% ethanol v/v mixed to water for 17 weeks. BMD was assessed by DXA, while the microarchitecture was analyzed using μCT. Bone remodeling was studied measuring serum concentration of osteocalcin, NTx and TRAP. Bone marrow adiposity, osteoblastic lineage differentiation, osteocyte morphology and apoptosis were assessed using bright field, epifluorescence, transmission electron and confocal microscopy. RESULTS BMD, trabecular thickness, TRAP and NTx concentration were significantly decreased in A35, while cortical thickness was thinner. There were 10 fold more cells stained with cleaved caspase-3, and 35% more empty lacunae in A35, these data indicating a large increase in osteocyte apoptosis in the A35 group. The number of lipid droplets in the marrow was increased in A35 (7 fold). Both the osteocyte apoptosis and the fat bone marrow content strongly correlated with femur BMD (p=0.0017, r = -0.72 and p=0.002, r = -0.70) and whole body BMD. CONCLUSION These data suggest that low BMD is associated with osteocyte apoptosis and bone marrow fat content in alcohol-induced osteopenia.

Osteocalcin-insulin relationship in obese children: a role for the skeleton in energy metabolism

Objective  Osteocalcin is a bone‐specific protein secreted by osteoblasts and often used as a bone formation biomarker. Rodent studies have reported a hormonal role of osteocalcin on glucose metabolism, increasing insulin secretion and sensitivity and increasing energy expenditure. However, it is unknown whether osteocalcin fulfils the same function in humans.

Regular exercise limits alcohol effects on trabecular, cortical thickness and porosity, and osteocyte apoptosis in the rat

INTRODUCTION Excessive alcohol consumption is known to be a cause of secondary osteoporosis whereas physical activity is recommended in prevention of osteoporosis. This study was designed to analyze the effects of physical exercise on bone parameters in chronic alcohol-fed rats. METHODS Forty-eight male Wistar rats were divided in four groups: Control (C), Alcohol (A), Exercise (E) and Alcohol+Exercise (AE). A and AE groups drank a solution composed of ethanol and water (35% volume/volume for 17 weeks). E and AE groups were submitted to treadmill training for 14 weeks (60 min/day, 5 times/week). Bone mineral density (BMD) was assessed by DXA, the trabecular and cortical microarchitectural parameters by microCT and serum osteocalcin, NTx and leptin concentrations by ELISA assays. Bone mechanical parameters were evaluated through mechanical testing. Osteocyte apoptosis was analyzed with cleaved caspase-3 immunostaining. RESULTS Alcohol-fed rats had significantly lower body weight (-28%), fat (-46%) and lean mass (-25%) compared to controls. BMD (-8%), trabecular (-12%) and cortical thickness (-27%) were significantly lower with alcohol whereas porosity (+38%) and pore number (+42%) were higher. Exercise combined with alcohol prevented lower Tb.Th (+20%), Ct.Th (+30%), stress (+26%) and higher Ct.Po (-24%) and osteocyte apoptosis (-91%) compared to A. However, WB BMD (-4%) and femur BMD were still lower in AE versus C. CONCLUSION Regular physical activity has beneficial effects on some microarchitectural parameters in alcohol-fed rats. However, regular treadmill exercise does not compensate for the effects of heavy chronic alcohol consumption on whole body bone density.

Synchrotron Ultraviolet Microspectroscopy on Rat Cortical Bone: Involvement of Tyrosine and Tryptophan in the Osteocyte and Its Environment

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately.

A Non-Destructive Method for Distinguishing Reindeer Antler (Rangifer tarandus) from Red Deer Antler (Cervus elaphus) Using X-Ray Micro-Tomography Coupled with SVM Classifiers

Over the last decade, biomedical 3D-imaging tools have gained widespread use in the analysis of prehistoric bone artefacts. While initial attempts to characterise the major categories used in osseous industry (i.e. bone, antler, and dentine/ivory) have been successful, the taxonomic determination of prehistoric artefacts remains to be investigated. The distinction between reindeer and red deer antler can be challenging, particularly in cases of anthropic and/or taphonomic modifications. In addition to the range of destructive physicochemical identification methods available (mass spectrometry, isotopic ratio, and DNA analysis), X-ray micro-tomography (micro-CT) provides convincing non-destructive 3D images and analyses. This paper presents the experimental protocol (sample scans, image processing, and statistical analysis) we have developed in order to identify modern and archaeological antler collections (from Isturitz, France). This original method is based on bone microstructure analysis combined with advanced statistical support vector machine (SVM) classifiers. A combination of six microarchitecture biomarkers (bone volume fraction, trabecular number, trabecular separation, trabecular thickness, trabecular bone pattern factor, and structure model index) were screened using micro-CT in order to characterise internal alveolar structure. Overall, reindeer alveoli presented a tighter mesh than red deer alveoli, and statistical analysis allowed us to distinguish archaeological antler by species with an accuracy of 96%, regardless of anatomical location on the antler. In conclusion, micro-CT combined with SVM classifiers proves to be a promising additional non-destructive method for antler identification, suitable for archaeological artefacts whose degree of human modification and cultural heritage or scientific value has previously made it impossible (tools, ornaments, etc.).

Alcohol‐induced Osteonecrosis – dose and duration effects

We read with great interest the paper by Ikemura et al. published in the August issue of the International Journal of Experimental Pathology (Ikemura et al. 2011). In their study, the authors used a model of alcohol-induced osteonecrosis in rabbits to evaluate the morphological changes in bone marrow fat cells and the changes in the serum lipid levels. Animals received either 15 ml/kg/day (LDA: low-dose alcohol) or 30 ml/kg/day (HDA: high-dose alcohol) of a solution containing 15% ethanol for intragastrically for 4 weeks, whereas a control group received a physiological saline solution. Results were observed at 6 weeks after the beginning of the treatment (i.e. 2 weeks after withdrawal). As these results are difficult to reconcile with aspects of current understanding of alcohol effects on bone metabolism, we would like to comment on their protocol and their results in perspective. Regarding the quantity of alcohol used to induce osteonecrosis, the animals thus ingested either 2.25 g/kg/day (LDA) or 4.5 g/kg/day (HDA) of alcohol. In comparison, Wang et al. (2008) used a dose of 9.2 g/kg/day (i.e. more than twice the amount used in the HDA group) to induce objectively recognizable alcohol-induced osteonecrosis in rabbits, with concomitant marked marrow fat cell hypertrophy and proliferation, thinner and sparse trabeculae, diminished hematopoiesis and increased empty osteocyte lacunae (Wang et al. 2008). In the same manner, Broulik et al.(2010) used a dose of 7.22 g/kg/day of ethanol (corresponding to a consumption of 1 l wine or 2.5 l of 12 ° beer in male adults) to induce bone metabolism disturbances in rats (Broulik et al. 2010). In animal models there may be a cause and effect relationship between alcohol consumption and osteonecrosis of the femoral head (Hirota et al. 1993), but for osteonecrosis to develop in humans, the alcohol exposure threshold is approximately 150 l of 100% ethanol, at a consumption rate of 400 ml or more of absolute ethanol weekly (Cruess 1986; Jones 1994). Therefore, we believe that it is difficult to compare rabbit, rat and human studies with regard to the quantity of alcohol used to induce bone disturbances. Bearing this in mind, we suggest that the lack of osteonecrotic lesions in both LDA and HDA groups might be linked to the fact that the doses of alcohol ingested over 4 weeks was too low a dose of alcohol. We believe that the authors need to justify and discuss their choice of dosage. In addition, the short duration of alcohol treatment (4 weeks) is surprising. The authors do not give an explanation for this duration of alcohol treatment. In comparison, alcohol was administered intragastrically to rabbits for one to 6 months, and changes in lipid metabolism were observed only after 2–3 months of treatment (Wang et al. 2003). In another study, mice were treated with alcohol daily for up to 10 months and significant modifications of lipid metabolism appeared at 6 months, with significant increase in largest fat cell size detectable at 10 months (Wang et al. 2008). Finally, animals were treated with alcohol for 4 weeks, and the results were observed at 6 weeks after the beginning of the treatment, therefore after 2 weeks of withdrawal. The authors do not discuss this aspect of the protocol and its relevance in the aetiology of induced osteonecrosis. A possible explanation is to mimic the clinically relevant symptoms of alcohol withdrawal (Schuckit 2009). However, enlightenment about the impact of this withdrawal on osteonecrosis development would be welcome. Thus, without affecting the reported results, we suggest that the findings are difficult to interpret based upon current understanding of alcohol effects on bone metabolism and that there are protocol-related issues, including dose, duration and eventual withdrawal, which might account for the observations reported.