Bone is a metabolically active tissue that is continuously remodeled throughout life. Osteocytes, the most abundant cells in bone, regulate bone homeostasis in response to hormonal and mechanical cues. Parathyroid hormone (PTH), a calciotropic hormone secreted from the parathyroid glands, has been widely used in the clinic to treat age-related osteoporosis. PTH acts on cells of the osteoblast lineage, including osteocytes, by signaling via the PTH receptor (PPR) to promote bone formation. However, the role of PPR signaling in osteocytes during aging has not been investigated. The hypothesis of this study is that PPR signaling in osteocytes plays a key role in maintaining skeletal health in aging mice. To address this hypothesis, mice in which the PPR was ablated in mature osteoblasts/osteocytes (Dmp1-Cre+;PPRfl/fl or Dmp1-PPRKO) were used to study their skeletal phenotype at 4 and 13 months of age. Compared to control littermates (Dmp1-Cre–;PPRfl/fl), Dmp1-PPRKO animals displayed age-dependent osteopenia due to reduced osteoblast activity and increased osteoclast numbers and activity. These changes were associated with a significant decrease in osteoprogenitors and an increase in marrow adiposity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes was accompanied by a marked increase in serum sclerostin, RANKL-expressing marrow adipocytes, and early onset of oxidative stress in osteocytes. In vitro studies demonstrated that PTH protected osteocytes from oxidative stress-induced cell death by suppressing the intracellular accumulation of reactive oxygen species. Mechanical forces are also important regulators of bone mass and quality. For instance, immobilization and reduced mechanical loading, such as prolonged bed rest or long-duration spaceflight, lead to bone loss or osteopenia due to reduced bone formation and increased bone resorption. Osteocytes are known to sense and transduce mechanical forces applied to the skeleton into biochemical signals. However, the exact molecular mechanism remains unclear. To unravel the mechanism by which osteocytes sense and respond to mechanical unloading, an osteocytic cell line, Ocy454, was exposed to microgravity (µG) conditions for 2, 4, or 6 days onboard the SpaceX Dragon-6 and the International Space Station. Global transcriptomic analysis demonstrated that µG leads to downregulation of key osteocytic marker genes compared to ground controls (1G), suggesting the impaired differentiation of osteocytes. Importantly, glycolysis was the most activated signaling pathway in osteocytes subjected to µG compared to 1G. Gene comparison analysis further identified a set of mechano-sensitive genes that are consistently regulated in multiple types of cells exposed to µG, suggesting a common, yet to be fully elucidated, genome-wide response to µG. In summary, these studies demonstrated that osteocytes are highly regulated by PTH and mechanical forces. We found that PPR signaling in osteocytes is important for protecting the skeleton from age-induced osteopenia by promoting osteoblast’s bone-forming activity and mitigating osteoclast’s bone resorption. We also demonstrated that PTH protects osteocytes from oxidative stress. Finally, we showed that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

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Introduction : Ce programme de recherche visait à étudier l'impact d'une intervention de 8 mois entrainant une perte de poids induite par l'activité physique et la nutrition sur la santé osseuse chez des adolescents obèses. L'objectif global de cette thèse était d'examiner l'impact d'une intervention de perte de poids sur les paramètres osseux chez les adolescents obèses.Méthode : Soixante-cinq adolescents ont été recrutés : 31 (6 garçons) obèses pour le groupe intervention (âge : 13,61 (1,27)), 23 adolescents de poids normal (NW) (âge : 15,90 (0,43)) et 11 (4 hommes) adolescents obèses pour le groupe témoin (14.02 (1.39)). Le critère d’évaluation principal concernait la densitométrie osseuse par DXA (corps entier, colonne vertébrale, hanche). Les critères d'évaluation secondaires comprenaient la composition corporelle (DXA), la géométrie et la résistance des os (analyse structurelle de la hanche) et des biomarqueurs osseux (propeptide N-terminal (P1NP) procollagène de type 1, estradiol C-télopeptide (CTx), leptine). Les données ont été collectées au départ à 4 mois et à 8 mois. Les données ont été ajustées en fonction des changements de poids corporel, de masse grasse et de masse maigre.Résultats : Comparés au groupe contrôle de poids normal, les adolescents obèses présentaient une densité osseuse non ajustée et ajustée inférieure. Suite à la perte de poids (~ -11%), les adolescents obèses ont augmentés leur densité osseuse au corps entier (% Ob 3,22 (3,58) p <0,001) et à la colonne lombaire (% Ob 6,27 (12,45) p = 0,014). Cependant, ces valeurs restent inférieures à celles de leurs homologues contrôle de poids normal après ajustement aux variations de poids corporel. Après l’intervention entrainant une perte de poids, les estimations du risque de fracture sont restées élevée, en particulier au niveau du col étroit (buckling ratio (BR) 8,25 (2,00) p = 0,005) et ce malgré des adaptations positives de certaines propriétés géométriques (i.e. NN CSA, NN Z). De plus, les modifications de l'accrétion osseuse chez les adolescents obèses suivent une adaptation de type androgènes, cela est démontrée par une expansion périostée (% NW ∆ 0,69 (3,71); Ob ∆ 1,67 (9,11)) et une résorption endocorticale (% NW ∆ -2,11 (11,79); Ob ∆ 4,42 (10,56)). Dans le groupe intervention, les différences au regard des marqueurs osseux favorisent la formation osseuse au cours des 4 premiers mois alors que par la suite la résorption osseuse est favorisée.Conclusion : La fragilité osseuse chez les adolescents obèses a été démontrée par (1) une densité minérale osseuse inférieure corps entier et régionale pré et post-intervention par rapport aux contrôles normo-pondérés, (2) un indice de risque de fracture élevé après intervention au niveau du cou étroit, (3) des biomarqueurs osseux démontrant des z-scores, indices de découplage (uncoupling indices) et représentations qualitatives de la distribution du remodelage osseux inférieures. Les résultats de cette thèse contribuent aux recherches futures sur les liens entre os et obésité à l’adolescence. / Introduction: This program of research targeted the impact of an 8-month weight loss intervention induced by physical activity and nutrition on bone health in adolescents with obesity. The overall aim of this thesis was to examine the impact of a lifestyle weight loss intervention on the bone parameters in adolescents with obesity. Method: Sixty-five adolescents were recruited: 31 (6 males) adolescents with obesity in the weight loss intervention (age: 13.61 (1.27)), 23 normal weight (NW) adolescents (age: 15.90 (0.43)) and 11 (4 males) adolescents with obesity in another control group (14.02 (1.39)). Primary outcomes targeted bone densitometry (whole body, spine, hip DXA). Secondary outcomes included body composition, bone geometry and strength (hip structural analysis) and bone biomarkers (procollagen type 1 N-terminal propeptide (P1NP), C telopeptide (CTx) estradiol, leptin). Data were collected at baseline, 4 months and 8 months. Data were adjusted for body weight, fat mass and lean mass changes.Results: Compared with the NW controls, adolescents with obesity displayed lower unadjusted and adjusted bone density. Following successful weight loss (~ -11%) adolescents with obesity increased whole body (%Ob ∆ 3.22 (3.58) p<0.001) and lumbar spine (%Ob ∆ 6.27 (12.45) p=0.014) BMD. However, values remain lower than their NW peers after adjustment to body weight changes. After the weight loss intervention, compromised estimates of fracture risk remained especially at the narrow neck (buckling ratio (BR) 8.25 (2.00) p=0.005), despite positive adaptations of some geometric properties (i.e. NN CSA, NN Z). Also, bone accretion changes in adolescents with obesity followed an androgen-like adaptation demonstrated by periosteal expansion (% NW ∆ 0.69 (3.71); Ob ∆ 1.67 (9.11)) and endocortical resorption (% NW ∆ -2.11 (11.79); Ob ∆ 4.42 (10.56)). Among the intervention group, differences in bone markers favoured formation during the first 4 months and favoured resorption in the remaining months.Conclusion: Bone fragility in adolescents with obesity was demonstrated by (1) baseline and post intervention lower whole body and regional BMD than NW controls, (2) post-intervention higher fracture risk index at the narrow neck, (3) bone biomarkers showing reduced z-scores, uncoupling indices and qualitative representations of the distribution of bone remodeling. Future investigations of links between bone and obesity during adolescence can be well informed by the results of this thesis.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.

An emerging non-pharmacological treatment for bone degenerative diseases is whole body vibration (WBV), a mechanical signal composed of low-magnitude, high-frequency (LMHF) vibrations that when applied to bone, have osteogenic and anti-resorptive effects. Currently, the cellular and molecular mechanism underlying the effect of WBV on bone is unclear. In this study, we investigated the response of osteocytes, the putative mechanosensor in bone, under LMHF vibration. As bone cells differentiate from mesenchymal stromal cells (MSCs), we also studied the osteogenic differentiation of rat MSCs in the presence of vibration loading. We found that vibrated osteocytes show gene and protein expression changes suggestive of an anti-osteoclastogenic response, and secrete soluble factors that inhibit osteoclast formation and activity. In contrast, rat MSCs showed moderate to no response to LMHF vibration during osteogenic differentiation. Our data suggest that in vivo effects of LMHF vibration are mediated through mechanosensing and biochemical responses by osteocytes.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

An emerging non-pharmacological treatment for bone degenerative diseases is whole body vibration (WBV), a mechanical signal composed of low-magnitude, high-frequency (LMHF) vibrations that when applied to bone, have osteogenic and anti-resorptive effects. Currently, the cellular and molecular mechanism underlying the effect of WBV on bone is unclear. In this study, we investigated the response of osteocytes, the putative mechanosensor in bone, under LMHF vibration. As bone cells differentiate from mesenchymal stromal cells (MSCs), we also studied the osteogenic differentiation of rat MSCs in the presence of vibration loading. We found that vibrated osteocytes show gene and protein expression changes suggestive of an anti-osteoclastogenic response, and secrete soluble factors that inhibit osteoclast formation and activity. In contrast, rat MSCs showed moderate to no response to LMHF vibration during osteogenic differentiation. Our data suggest that in vivo effects of LMHF vibration are mediated through mechanosensing and biochemical responses by osteocytes.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.