Connexin 43 and cell culture substrate differentially regulate OCY454 osteocytic differentiation and signaling to primary bone cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests that osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption; however, the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using three-dimensional (3D) culture substrates in OCY454 cells suggest that 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and receptor activator of nuclear factor-κB ligand (RANKL). In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds with traditional 2D tissue culture, both with [wild-type (WT)] and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures were used to determine soluble signaling to differentiate primary bone marrow cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared with that of Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion.NEW & NOTEWORTHY 3D cell culture of OCY454 cells promoted increased differentiation compared with traditional 2D culture. Although Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Connexin 43 and Cell Culture Substrate Differentially Regulate OCY454 Osteocytic Differentiation and Signaling to Primary Bone Cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption, however the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using 3D culture substrates in OCY454 cells suggest 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and RANKL. In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds to traditional 2D tissue culture, both with (WT) and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures was used to determine soluble signaling to differentiate primary bone marrow stromal cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared to Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and increased osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion. New and Noteworthy: 3D cell culture of OCY454 cells promoted increased differentiation compared to traditional 2D culture. While Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Similarities Between Disuse and Age-Induced Bone Loss.

Disuse and aging are known risk factors associated with low bone mass and quality deterioration, resulting in increased fracture risk. Indeed, current and emerging evidence implicate a large number of shared skeletal manifestations between disuse and aging scenarios. This review provides a detailed overview of current preclinical models of musculoskeletal disuse and the clinical scenarios they seek to recapitulate. We also explore and summarize the major similarities between bone loss after extreme disuse and advanced aging at multiple length scales, including at the organ/tissue, cellular, and molecular level. Specifically, shared structural and material alterations of bone loss are presented between disuse and aging, including preferential loss of bone at cancellous sites, cortical thinning, and loss of bone strength due to enhanced fragility. At the cellular level bone loss is accompanied, during disuse and aging, by increased bone resorption, decreased formation, and enhanced adipogenesis due to altered gap junction intercellular communication, WNT/ß-catenin and RANKL/OPG signaling. Major differences between extreme short-term disuse and aging are discussed, including anatomical specificity, differences in bone turnover rates, periosteal modeling, and the influence of subject sex and genetic variability. The examination also identifies potential shared mechanisms underlying bone loss in aging and disuse that warrant further study such as collagen cross-linking, advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling, reactive oxygen species (ROS) and nuclear factor κB (NF-κB) signaling, cellular senescence, and altered lacunar-canalicular connectivity (mechanosensation). Understanding the shared structural alterations, changes in bone cell function, and molecular mechanisms common to both extreme disuse and aging are paramount to discovering therapies to combat both age-related and disuse-induced osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Stable Encapsulation of Air in Mesoporous Silica Nanoparticles: Fluorocarbon-Free Nanoscale Ultrasound Contrast Agents.

While gas-filled micrometer-sized ultrasound contrast agents vastly improve signal-to-noise ratios, microbubbles have short circulation lifetimes and poor extravasation from the blood. Previously reported fluorocarbon-based nanoscale contrast agents are more stable but their contrast is generally lower owing to their size and dispersity. The contrast agents reported here are composed of silica nanoparticles of ≈100 nm diameter that are filled with ≈3 nm columnar mesopores. Functionalization of the silica surface with octyl groups and resuspension with Pluronic F127 create particles with pores that remain filled with air but are stable in buffer and serum. Administration of high intensity focused ultrasound (HIFU) allows sensitive imaging of the silica nanoparticles down to 10(10) particles mL(-1) , with continuous imaging for at least 20 min. Control experiments with different silica particles supported the hypothesis that entrapped air could be pulled into bubble nuclei, which can then in turn act as acoustic scatterers. This process results in very little hemolysis in whole blood, indicating potential for nontoxic blood pool imaging. Finally, the particles are lyophilized and reconstituted or stored in PBS (phosphate-buffered saline, at least for four months) with no loss in contrast, indicating stability to storage and reformulation.

Phase Behavior of Mixed Lipid Monolayers on Perfluorocarbon Nanoemulsions and its Effect on Acoustic Contrast.

Lipid-stabilized nanoemulsions containing a volatile liquid perfluorocarbon core have been studied as ultrasound contrast agents owing to their ability to transform into high-contrast microbubbles when subjected to high intensity focused ultrasound (HIFU). However, while there have been several studies on the effect of acoustic parameters on contrast, the effect of the droplet's stabilizing shell has not been studied as extensively. Inspired by previous studies showing lateral phase separation in microbubbles and vesicles, nanodroplets were formulated with a perfluorohexane core and a shell composed of varying amounts of saturated (DPPC) phospholipids, unsaturated (DOPC) phospholipids, and cholesterol, which were fractionated to obtain nanodroplets of mean diameter 300-400 nm and were stable over one week. When the DOPC content was increased to 40 mol%, ultrasound contrast increased by about one order of magnitude over DPPC-only droplets. Based on fluorescence microscopy results of lateral lipid phase separation on the droplet surface, the various combinations of DPPC, DOPC, and cholesterol were assigned to three regimes on the ternary phase diagram: solid-liquid ordered (low contrast), liquid ordered-liquid disordered (medium contrast), and solid-liquid disordered (high contrast). These regimes were confirmed by TEM analysis of nanoscale droplets. Droplets containing mixed lipid monolayers were also found to produce a significantly greater yield than single-component droplets. The discovery of the dependence of acoustic response on lipid phase separation will help to understand the formulation, behavior, and vaporization mechanism of acoustically-responsive nanoemulsions.