Computational modeling based on confocal imaging predicts changes in osteocyte and dendrite shear stress due to canalicular loss with aging.

Exercise and physical activity exert mechanical loading on the bones which induces bone formation. However, the relationship between the osteocyte lacunar-canalicular morphology and mechanical stress experienced locally by osteocytes transducing signals for bone formation is not fully understood. In this study, we used computational modeling to predict the effect of canalicular density, the number of fluid inlets, and load direction on fluid flow shear stress (FFSS) and bone strains and how these might change following the microstructural deterioration of the lacunar-canalicular network that occurs with aging. Four distinct computational models were initially generated of osteocytes with either ten or eighteen dendrites using a fluid-structure interaction method with idealized geometries. Next, a young and a simulated aged osteocyte were developed from confocal images after FITC staining of the femur of a 4-month-old C57BL/6 mouse to estimate FFSS using a computational fluid dynamics approach. The models predicted higher fluid velocities in the canaliculi versus the lacunae. Comparison of idealized models with five versus one fluid inlet indicated that with four more inlets, one-half of the dendrites experienced FFSS greater than 0.8 Pa, which has been associated with osteogenic responses. Confocal image-based models of real osteocytes indicated a six times higher ratio of canalicular to lacunar surface area in the young osteocyte model than the simulated aged model and the average FFSS in the young model (FFSS = 0.46 Pa) was three times greater than the aged model (FFSS = 0.15 Pa). Interestingly, the surface area with FFSS values above 0.8 Pa was 23 times greater in the young versus the simulated aged model. These findings may explain the impaired mechano-responsiveness of osteocytes with aging.

Osteocyte RANKL Drives Bone Resorption in Mouse Ligature-Induced Periodontitis.

Mouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Body weight influences musculoskeletal adaptation to long-term voluntary wheel running during aging in female mice.

Frailty is the hallmark of aging that can be delayed with exercise. The present studies were initiated based on the hypothesis that long-term voluntary wheel running (VWR) in female mice from 12 to 18 or 22 months of age would have beneficial effects on the musculoskeletal system. Mice were separated into high (HBW) and low (LBW) body weight based on final body weights upon termination of experiments. Bone marrow fat was significantly higher in HBW than LBW under sedentary conditions, but not with VWR. HBW was more protective for soleus size and function than LBW under sedentary conditions, however VWR increased soleus size and function regardless of body weight. VWR plus HBW was more protective against muscle loss with aging. Similar effects of VWR plus HBW were observed with the extensor digitorum longus, EDL, however, LBW with VWR was beneficial in improving EDL fatigue resistance in 18 mo mice and was more beneficial with regards to muscle production of bone protective factors. VWR plus HBW maintained bone in aged animals. In summary, HBW had a more beneficial effect on muscle and bone with aging especially in combination with exercise. These effects were independent of bone marrow fat, suggesting that intrinsic musculoskeletal adaptions were responsible for these beneficial effects.

Assessment of Osteocytes: Techniques for Studying Morphological and Molecular Changes Associated with Perilacunar/Canalicular Remodeling of the Bone Matrix.

Recent advances have revived interest in the concept of osteocyte perilacunar/canalicular remodeling (PLR) and have motivated efforts to identify the mechanisms regulating this process in bone in the context of normal physiology and pathological conditions. Here, we describe several methods that are evaluating morphological changes associated with PLR function of osteocytes.

Using confocal imaging approaches to understand the structure and function of osteocytes and the lacunocanalicular network.

Although overlooked in the past, osteocytes have come to the forefront of skeletal biology and are now recognized as a key cell type that integrates hormonal, mechanical and other signals to control bone mass through regulation of both osteoblast and osteoclast activity. With the surge of recent interest in osteocytes as bone regulatory cells and the discovery that they also function as endocrine regulators of phosphate homeostasis, there has been renewed interest in understanding the structure and function of these unique and relatively inaccessible cells. Osteocytes are embedded within the mineralized bone matrix and are housed within a complex lacunocanalicular system which connects them with the circulation and with other organ systems. This has presented unique challenges for imaging these cells. This review summarizes recent advances in confocal imaging approaches for visualizing osteocytes and their lacunocanalicular networks in both living and fixed bone specimens and discusses how computational approaches can be combined with live and fixed cell imaging techniques to generate quantitative outputs and predictive models. The integration of advanced imaging with computational approaches promises to lead to a more in depth understanding of the structure and function of osteocyte networks and the lacunocanalicular system in the healthy and aging state as well as in pathological conditions in bone.

Osteocyte lacunar strain determination using multiscale finite element analysis.

Osteocytes are thought to be the primary mechanosensory cells within bone, regulating both osteoclasts and osteoblasts to control load induced changes in bone resorption and formation. Osteocytes initiate intracellular responses including activating the Wnt/ß-catenin signaling pathway after experiencing mechanical forces. In response to changing mechanical loads (strain) the osteocytes signal to cells on the bone surface. However, this process of osteocyte activation appears heterogeneous since it occurs in sub-populations of osteocytes, even within regions predicted to be experiencing similar global strain magnitudes determined based on traditional finite element modeling approaches. Several studies have investigated the strain responses of osteocyte lacunae using finite element (FE) models, but many were limited by the use of idealized geometries (e.g., ellipsoids) and analysis of a single osteocyte. Finite element models by other groups included more details, such as canaliculi, but all were done on models consisting of a single osteocyte. We hypothesized that variation in size and orientation of the osteocyte lacunae within bone would give rise to micro heterogeneity in the strain fields that could better explain the observed patterns of osteocyte activation following load. The osteocytes in our microscale and nanoscale models have an idealized oval shape and some are based on confocal scans. However, all the FE models in this preliminary study consist of multiple osteocytes. The number of osteocytes in the 3D confocal scan models ranged from five to seventeen. In this study, a multi-scale computational approach was used to first create an osteocyte FE model at the microscale level to examine both the theoretical lacunar and perilacunar strain responses based on two parameters: 1) lacunar orientation and 2) lacunar size. A parametric analysis was performed by steadily increasing the perilacunar modulus (5, 10, 15, and 20 GPa). Secondly, a nanoscale FE model was built using known osteocyte dimensions to determine the predicted strains in the perilacunar matrix, fluid space, and cell body regions. Finally, 3-D lacunar models were created using confocal image stacks from mouse femurs to determine the theoretical strain in the lacunae represented by realistic geometries. Overall, lacunar strains decreased by 14% in the cell body, 15% in the fluid space region and 25% in the perilacunar space as the perilacunar modulus increased, indicating a stress shielding effect. Lacunar strains were lower for the osteocytes aligned along the loading axis compared to those aligned perpendicular to axis. Increases in lacuna size also led to increased lacunar strains. These finite element model findings suggest that orientation and lacunar size may contribute to the heterogeneous initial pattern of osteocyte strain response observed in bone following in vivo applied mechanical loads. A better understanding of how mechanical stimuli directly affect the lacunae and perilacunar tissue strains may ultimately lead to a better understanding of the process of osteocyte activation in response to mechanical loading.

Age-dependent regulation of cell-mediated collagen turnover.

Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

Exosomes and Extracellular RNA in Muscle and Bone Aging and Crosstalk.

PURPOSE OF REVIEW: Extracellular vesicles (EV), which include exosomes and microvesicles, are membrane-bound particles shed by most cell types and are important mediators of cell-cell communication by delivering their cargo of proteins, miRNA, and mRNA to target cells and altering their function. Here, we provide an overview of what is currently known about EV composition and function in bone and muscle cells and discuss their role in mediating crosstalk between these two tissues as well as their role in musculoskeletal aging. RECENT FINDINGS: Recent studies have shown that muscle and bone cells produce EV, whose protein, mRNA, and miRNA cargo reflects the differentiated state of the parental cells. These EV have functional effects within their respective tissues, but evidence is accumulating that they are also shed into the circulation and can have effects on distant tissues. Bone- and muscle-derived EV can alter the differentiation and function of bone and muscle cells. Many of these effects are mediated via small microRNAs that regulate target genes in recipient cells. EV-mediated signaling in muscle and bone is an exciting and emerging field. While considerable progress has been made, much is still to be discovered about the mechanisms regulating EV composition, release, uptake, and function in muscle and bone. A key challenge is to understand more precisely how exosomes function in truly physiological settings.

Fibroblast growth factor 9 (FGF9) inhibits myogenic differentiation of C2C12 and human muscle cells.

Osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. The biochemical crosstalk between muscle and bone seems to play a role in OS. We have previously shown that osteocytes produce soluble factors with beneficial effects on muscle and vice versa. Recently, enhanced FGF9 production was observed in the OmGFP66 osteogenic cell line. To test its role in myogenic differentiation, C2C12 myoblasts were treated with recombinant FGF9. FGF9 as low as 10 ng/mL inhibited myogenic differentiation, suggesting that FGF9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. FGF9 (10-50 ng/mL) significantly decreased mRNA expression of MyoG and Mhc while increasing the expression of Myostatin. Consistent with the phenotype, RT-qPCR array revealed that FGF9 (10 ng/mL) increased the expression of Icam1 while decreased the expression of Wnt1 and Wnt6 decreased, respectively. FGF9 decreased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and reduced the expression of genes (i.e. Cacna1s, RyR2, Naftc3) directly associated with intracellular Ca2+ homeostasis. Myogenic differentiation in human skeletal muscle cells was similarly inhibited by FGF9 but required higher doses of 200 ng/mL FGF9. FGF9 was also shown to stimulate C2C12 myoblast proliferation. FGF2 and the FGF9 subfamily members FGF16 and FGF20 also inhibited C2C12 myoblast differentiation and enhanced proliferation. Intriguingly, the differentiation inhibition was independent of proliferation enhancement. These findings suggest that FGF9 may modulate myogenesis via a complex signaling mechanism.

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization.

Bone formation, remodeling and repair are dynamic processes, involving cell migration, ECM assembly, osteocyte embedding, and bone resorption. Using live-cell imaging, we previously showed that osteoblast assembly of the ECM proteins fibronectin and collagen is highly dynamic and is integrated with cell motility. Additionally, osteoblast-to-osteocyte transition involved arrest of cell motility, followed by dendrite extension and retraction that may regulate positioning of embedding osteocytes. To further understand how osteocytes differentiate and embed in collagen, mice were generated that co-expressed GFPtopaz-tagged collagen with a Dmp1-Cre-inducible tdTomato reporter targeted to preosteocytes/osteocytes. Dual live-cell imaging of collagen and osteocyte dynamics in mineralizing primary calvarial cell cultures showed that Dmp1-Cre/tdTomato turned on in early bone nodule forming regions, demarcated by foci of concentrated GFP-collagen bundles that appeared structurally distinct from the surrounding collagen. Dmp1-Cre/tdTomato-positive cells were post-mitotic and were continuously induced throughout the 2 week timecourse, whereas the majority of collagen was assembled by day 7. GFP-collagen fibrils showed global (tissue-level) motions, suggesting coordinated cell layer movement, and local fibril motions mediated by cell-generated forces. Condensation of collagen fibril networks occurred within bone nodules prior to mineralization. Intravital imaging confirmed a similar structural appearance of GFP-collagen in calvarial bone, with analogous global motions of mineralizing areas adjacent to sutures. In early (unmineralized) calvarial cell cultures, Dmp1-Cre/tdTomato-positive cells were motile (mean velocity 4.8 µm/h), moving freely in and around the forming bone nodule, with a small number of these cells embedded in collagen, constraining their motion. In mineralizing cultures, the average velocity of Dmp1-Cre/tdTomato-positive cells was significantly reduced (0.7 µm/h), with many immobilized in the mineralizing nodule. Three apparent mechanisms for embedding of Dmp1-Cre/tdTomato-positive cells were observed. In some cases, a previously motile Dmp1-Cre/tdTomato-positive cell became immobilized in collagen fibril networks that were newly assembled around the cell, thereby entrapping it. In other cases, a motile Dmp1-Cre/tdTomato-positive cell moved into an already formed "collagen lacuna," arrested its motility and became embedded. Alternatively, some cells switched on tdTomato expression in situ within a lacuna. These data provide new insight into the dynamic process of bone collagen assembly and suggest multiple mechanisms for osteocyte entrapment in collagen matrix.

A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure.

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment. © 2019 American Society for Bone and Mineral Research.

Characterization of a novel murine Sost ERT2 Cre model targeting osteocytes.

Transgenic mice are widely used to delete or overexpress genes in a cell specific manner to advance knowledge of bone biology, function and disease. While numerous Cre models exist to target gene recombination in osteoblasts and osteoclasts, few target osteocytes specifically, particularly mature osteocytes. Our goal was to create a spatial and temporal conditional Cre model using tamoxifen to induce Cre activity in mature osteocytes using a Bac construct containing the 5' and 3' regions of the Sost gene (Sost ERT2 Cre). Four founder lines were crossed with the Ai9 Cre reporter mice. One founder line showed high and specific activity in mature osteocytes. Bones and organs were imaged and fluorescent signal quantitated. While no activity was observed in 2 day old pups, by 2 months of age some osteocytes were positive as osteocyte Cre activity became spontaneous or 'leaky' with age. The percentage of positive osteocytes increased following tamoxifen injection, especially in males, with 43% to 95% positive cells compared to 19% to 32% in females. No signal was observed in any bone surface cell, bone marrow, nor in muscle with or without tamoxifen injection. No spontaneous signal was observed in any other organ. However, with tamoxifen injection, a few positive cells were observed in kidney, eye, lung, heart and brain. All other organs, 28 in total, were negative with tamoxifen injection. However, with age, a muscle phenotype was apparent in the Sost-ERT2 Cre mice. Therefore, although this mouse model may be useful for targeting gene deletion or expression to mature osteocytes, the muscle phenotype may restrict the use of this model to specific applications and should be considered when interpreting data.

Changes in the osteocyte lacunocanalicular network with aging.

Osteoporosis is an aging-related disease of reduced bone mass that is particularly prevalent in post-menopausal women, but also affects the aged male population and is associated with increased fracture risk. Osteoporosis is the result of an imbalance whereby bone formation by osteoblasts no longer keeps pace with resorption of bone by osteoclasts. Osteocytes are the most abundant cells in bone and, although previously thought to be quiescent, they are now known to be active, multifunctional cells that play a key role in the maintenance of bone mass by regulating both osteoblast and osteoclast activity. They are also thought to regulate bone mass through their role as mechanoresponsive cells in bone that coordinate adaptive responses to mechanical loading. Osteocytes form an extensive interconnected network throughout the mineralized bone matrix and receive their nutrients as well as hormones and signaling factors through the lacunocanalicular system. Several studies have shown that the extent and connectivity of the lacunocanalicular system and osteocyte networks degenerates in aged humans as well as in animal models of aging. It is also known that the bone anabolic response to loading is decreased with aging. This review summarizes recent research on the degenerative changes that occur in osteocytes and their lacunocanalicular system as a result of aging and discusses the implications for skeletal health and homeostasis as well as potential mechanisms that may underlie these degenerative changes. Since osteocytes are such key regulators of skeletal homeostasis, maintaining the health of the osteocyte network would seem critical for maintenance of bone health. Therefore, a more complete understanding of the structure and function of the osteocyte network, its lacunocanalicular system, and the degenerative changes that occur with aging should lead to advances in our understanding of age related bone loss and potentially lead to improved therapies.

Live Cell Imaging of Bone Cell and Organ Cultures.

Over the past two decades there have been unprecedented advances in the capabilities for live cell imaging using light and confocal microscopy. Together with the discovery of green fluorescent protein and its derivatives and the development of a vast array of fluorescent imaging probes and conjugates, it is now possible to image virtually any intracellular or extracellular protein or structure. Traditional static imaging of fixed bone cells and tissues takes a snapshot view of events at a specific time point, but can often miss the dynamic aspects of the events being investigated. This chapter provides an overview of the application of live cell imaging approaches for the study of bone cells and bone organ cultures. Rather than emphasizing technical aspects of the imaging equipment, which may vary in different laboratories, we focus on what we consider to be the important principles that are of most practical use for an investigator setting up these techniques in their own laboratory. We also provide detailed protocols that our laboratory has used for live imaging of bone cell and organ cultures.

Mouse Cre Models for the Study of Bone Diseases.

PURPOSE: Transgenic Cre lines are a valuable tool for conditionally inactivating or activating genes to understand their function. Here, we provide an overview of Cre transgenic models used for studying gene function in bone cells and discuss their advantages and limitations, with particular emphasis on Cre lines used for studying osteocyte and osteoclast function. RECENT FINDINGS: Recent studies have shown that many bone cell-targeted Cre models are not as specific as originally thought. To ensure accurate data interpretation, it is important for investigators to test for unexpected recombination events due to transient expression of Cre recombinase during development or in precursor cells and to be aware of the potential for germ line recombination of targeted genes as well as the potential for unexpected phenotypes due to the Cre transgene. Although many of the bone-targeted Cre-deleter strains are imperfect and each model has its own limitations, their careful use will continue to provide key advances in our understanding of bone cell function in health and disease.

Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease.

Skeletal Response to Soluble Activin Receptor Type IIB in Mouse Models of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder primarily due to mutations in the type I collagen genes (COL1A1 and COL1A2), leading to compromised biomechanical integrity in type I collagen-containing tissues such as bone. Bone is inherently mechanosensitive and thus responds and adapts to external stimuli, such as muscle mass and contractile strength, to alter its mass and shape. Myostatin, a member of the TGF-ß superfamily, signals through activin receptor type IIB to negatively regulate muscle fiber growth. Because of the positive impact of myostatin deficiency on bone mass, we utilized a soluble activin receptor type IIB-mFc (sActRIIB-mFc) fusion protein in two molecularly distinct OI mouse models (G610C and oim) and evaluated their bone properties. Wild-type (WT), +/G610C, and oim/oim mice were treated from 2 to 4 months of age with either vehicle (Tris-buffered saline) or sActRIIB-mFc (10 mg/kg). Femurs of sActRIIB-mFc-treated mice exhibited increased trabecular bone volume regardless of genotype, whereas the cortical bone microarchitecture and biomechanical strength were only improved in WT and +/G610C mice. Dynamic histomorphometric analyses suggest the improved cortical bone geometry and biomechanical integrity reflect an anabolic effect due to increased mineral apposition and bone formation rates, whereas static histomorphometric analyses supported sActRIIB-mFc treatment also having an anti-catabolic impact with decreased osteoclast number per bone surface on trabecular bone regardless of sex and genotype. Together, our data suggest that sActRIIB-mFc may provide a new therapeutic direction to improve both bone and muscle properties in OI. © 2018 American Society for Bone and Mineral Research.

Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility.

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research.