Search

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix.

Osteophytes - bony outgrowths on joint structures - are found in healthy individuals but are specifically present in late osteoarthritis (OA). Osteophyte development and function is not well understood, yet biomechanical stimuli are thought to be critical. Bone adapts to mechanical forces via the cellular network of osteocytes. The involvement of osteocytes in osteophyte formation and maturation has not been unravelled. Forty-three osteophytes from tibias of 23 OA patients (65 ± 9 years) were analysed. The trabecular bone structure of osteophytes presented with fewer trabeculae of lower bone mineral density compared to subchondral bone. We identified 40% early stage and 60% late stage osteophytes that significantly differed in their trabecular bone characteristics. Osteophyte bone revealed a higher number of osteocytes and a lower number of empty osteocyte lacunae per bone area than the subchondral bone. We found that OA osteophytes consist of younger bone material comprised of woven and lamellar bone with the capacity to develop into a late stage osteophyte potentially via the involvement of the osteocyte network. Our analysis of OA osteophytes implies a transition from woven to lamellar bone as in physiological bone growth within a pathological joint. Therefore, osteophyte development and growth present a valuable research subject when aiming to investigate the osteogenic signalling cascade.

Osteoporosis is caused by increased bone resorption and decreased bone formation. Intermittent administration of a fragment of Parathyroid hormone (PTH) activates osteoblast-mediated bone formation and is used in patients with severe osteoporosis. However, the mechanisms by which PTH elicits its anabolic effect are not fully elucidated. Here we show that the absence of the homeodomain protein TG-interacting factor 1 (Tgif1) impairs osteoblast differentiation and activity, leading to a reduced bone formation. Deletion of Tgif1 in osteoblasts and osteocytes decreases bone resorption due to an increased secretion of Semaphorin 3E (Sema3E), an osteoclast-inhibiting factor. Tgif1 is a PTH target gene and PTH treatment failed to increase bone formation and bone mass in Tgif1-deficient mice. Thus, our study identifies Tgif1 as a novel regulator of bone remodeling and an essential component of the PTH anabolic action. These insights contribute to a better understanding of bone metabolism and the anabolic function of PTH.

Osteocytes, the most abundant bone cell in the adult skeleton, can function as mechanosensors directing osteoblast and osteoclast function in order to maintain optimal load bearing bone in addition to functioning as endocrine cells regulating phosphate metabolism. A controversial function, previously overlooked or denied, has been osteocytes as regulators of calcium metabolism. Early histologists upon observing enlarged osteocyte lacunae in bone sections proposed that mature osteocytes could remove their perilacunar matrix, a term called "osteocytic osteolysis". New insights into this process have occurred during the last decade using novel technology thereby providing a means to identify molecular mechanisms responsible for osteocytic osteolysis. As release of calcium from a mineralized matrix requires a more acidic pH and specialized enzymes, it was proposed that osteocytes may utilize similar molecular mechanisms as osteoclasts to remove mineral. The idea that a cell descended from mesenchymal progenitors (the osteocyte) could function similarly to a cell descended from hematopoietic progenitors (the osteoclast) was challenged as being improbable. Here we review the molecular mechanisms behind this osteocyte function, the role of osteocytic osteolysis in health and disease, and the capacity of the osteocyte to reverse the osteolytic process by replacing the removed matrix, a revived osteoblast function.

Excessive skeletal deformations and brittle fractures in the vast majority of patients suffering from osteogenesis imperfecta (OI) are a result of substantially reduced bone quality. Because the mechanical competence of bone is dependent on the tissue characteristics at small length scales, it is of crucial importance to assess how OI manifests at the micro- and nanoscale of bone. In this context, the Chihuahua (Chi/+) zebrafish, carrying a heterozygous glycine substitution in the α1 chain of collagen type I, has recently been proposed as a suitable animal model of classical dominant OI, showing skeletal deformities, altered mineralization patterns, and a smaller body size. This study assessed the bone quality properties of Chi/+ at multiple length scales using micro-computed tomography (micro-CT), histomorphometry, quantitative back-scattered electron imaging, Fourier-transform infrared spectroscopy, nanoindentation, and X-ray microscopy. At the skeletal level, the Chi/+ displays smaller body size, deformities, and fracture calli in the ribs. Morphological changes at the whole bone level showed that the vertebrae in Chi/+ had a smaller size, smaller thickness, and distorted shape. At the tissue level, Chi/+ displayed a higher degree of mineralization, lower collagen maturity, lower mineral maturity, altered osteoblast morphology, and lower osteocyte lacunar density compared to wild-type zebrafish. The alterations in the cellular, compositional, and structural properties of Chi/+ bones bear an explanation for the impaired local mechanical properties, which promote an increase in overall bone fragility in Chi/+. The quantitative assessment of bone quality in Chi/+ thus further validates this mutant as an important model reflecting osseous characteristics associated with human classical dominant OI. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFβ₃. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ₃ maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFβ₃ on co-cultured cells were serum-dependent. Also, TGFβ₃ reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFβ₃ maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). l-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Within the mineralized bone, osteocytes form a multifunctional mechanosensitive network orchestrating bone remodelling. A preserved osteocyte population is a crucial determinant of bone quality. In human auditory ossicles, the early decrease in osteocyte numbers but maintained integrity remains an unexplained phenomenon that might serve for sound transmission from air to the labyrinth. Here we analysed the frequency, size and composition of osteocyte lacunae in the auditory ossicles of 22 individuals from early postnatal period to old age. Mineralization of the bone matrix was determined using backscattered electron imaging. No signs of bone remodelling were observed above the age of 1 year. We detected characteristics of early bone tissue aging, such as decrease in osteocytes, lower total lacunar density and lacunar area, as well as high matrix mineralization accompanied by distinct accumulation of micropetrotic lacunae and decreased indentation depths. The majority of these changes took place in the first months and years of life, while afterwards only minor reorganization was present. With osteocyte apoptosis potentially being a consequence of low mechanical stimuli, the early loss of osteocytes without initiation of bone remodelling indicates an adaptive response conserving the architecture of the auditory ossicles and ensuring stable sound transmission throughout life.

Osteoporosis and sarcopenia are age-related musculoskeletal pathologies that often develop in parallel. Osteoporosis is characterized by a reduced bone mass and an increased fracture risk. Sarcopenia describes muscle wasting with an increasing risk of injuries due to falls. The medical treatment of both diseases costs billions in health care per year. With the impact on public health and economy, and considering the increasing life expectancy of populations, more efficient treatment regimens are sought. The biomechanical interaction between both tissues with muscle acting on bone is well established. Recently, both tissues were also determined as secretory endocrine organs affecting the function of one another. New exciting discoveries on this front are made each year, with novel signaling molecules being discovered and potential controversies being described. While this review does not claim completeness, it will summarize the current knowledge on both the biomechanical and the biochemical link between muscle and bone. The review will highlight the known secreted molecules by both tissues affecting the other and finish with an outlook on novel therapeutics that could emerge from these discoveries., Competing Interests: The authors declare no conflict of interest.

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1 . We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca 2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca 2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo , osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca 2+ signaling and the Wnt/β-Catenin pathway., Competing Interests: Disclosures: All authors state that they have no conflicts of interest.

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG-SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno-canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)-tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP-fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Unlabelled: Intramedullary stabilization is frequently used to treat long bone fractures. Implants usually remain unless complications arise. Since implant removal can become technically very challenging with the potential to cause further tissue damage, biodegradable materials are emerging as alternative options. Magnesium (Mg)-based biodegradable implants have a controllable degradation rate and good tissue compatibility, which makes them attractive for musculoskeletal research. Here we report for the first time the implantation of intramedullary nails made of an Mg alloy containing 2% silver (Mg2Ag) into intact and fractured femora of mice. Prior in vitro analyses revealed an inhibitory effect of Mg2Ag degradation products on osteoclast differentiation and function with no impair of osteoblast function. In vivo, Mg2Ag implants degraded under non-fracture and fracture conditions within 210days and 133days, respectively. During fracture repair, osteoblast function and subsequent bone formation were enhanced, while osteoclast activity and bone resorption were decreased, leading to an augmented callus formation. We observed a widening of the femoral shaft under steady state and regenerating conditions, which was at least in part due to an uncoupled bone remodeling. However, Mg2Ag implants did not cause any systemic adverse effects. These data suggest that Mg2Ag implants might be promising for intramedullary fixation of long bone fractures, a novel concept that has to be further investigated in future studies., Statement of Significance: Biodegradable implants are promising alternatives to standard steel or titanium implants to avoid implant removal after fracture healing. We therefore developed an intramedullary nail using a novel biodegradable magnesium-silver-alloy (Mg2Ag) and investigated the in vitro and in vivo effects of the implants on bone remodeling under steady state and fracture healing conditions in mice. Our results demonstrate that intramedullary Mg2Ag nails degrade in vivo over time without causing adverse effects. Importantly, radiographs, μCT and bone histomorphometry revealed a significant increase in callus size due to an augmented bone formation rate and a reduced bone resorption in fractures supported by Mg2Ag nails, thereby improving bone healing. Thus, intramedullary Mg2Ag nails are promising biomaterials for fracture healing to circumvent implant removal., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, that returned to virgin levels upon weaning. Infusion of parathyroid hormone-related peptide (PTHrP), known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the parathyroid hormone type 1 receptor (PTHR1) in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts., (Copyright © 2012 American Society for Bone and Mineral Research.)

In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

Background: Bone graft studies lack standardized controls. We aim to present a quick and reliable method for the intra-operative generation of acellular bone explants., Methods: Therefore, ovine cancellous bone explants from the iliac crest were prepared and used to test several methods for the induction of cell death. Over night heat inactivation was used as positive treatment control, methods to be investigated included UV light, or X- ray exposure, incubation in a hypotonic solution (salt-free water) and a short cycle of repeated freezing and thawing., Results: Viability of treated and 2 days cultured bone explants was investigated by lactate dehydrogenase assay. Non-treated cultured control explants maintained around 50% osteocyte viability, while osteocyte survival after the positive treatment control was abolished. The most dramatic loss in cell viability, together with a low standard deviation, was a repeated cycle of freezing and thawing., Conclusions: To summarize, we present a freeze-thaw method for the creation of acellular bone explants, which is easy to perform, not time-consuming and provides consistent results.