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Abstract Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Contrast-enhanced computed tomography offers a nondestructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using contrast-enhanced computed tomography but fail to provide chemical information. This study introduces isotonic Lugol’s iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae and bovine muscle tissue strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte gray values post-IL staining were associated with chemical distinctions between bovine muscle tissue and murine caudal vertebrae. More specific, a difference in the degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of gray value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.

Abstract Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.

The kidney’s microstructure, which comprises a highly convoluted tubular and vascular network, can only be partially revealed using classical 2D histology. Considering that the kidney’s microstructure is closely related to its function and is often affected by pathologies, there is a need for powerful and high-resolution 3D imaging techniques to visualize the microstructure. Here, we present how cryogenic contrast-enhanced microCT (cryo-CECT) allowed 3D visualization of glomeruli, tubuli, and vasculature. By comparing different contrast-enhancing staining agents and freezing protocols, we found that the preferred sample preparation protocol was the combination of staining with 1:2 hafnium(IV)-substituted Wells-Dawson polyoxometalate and freezing by submersion in isopentane at −78°C. This optimized protocol showed to be highly sensitive, allowing to detect small pathology-induced microstructural changes in a mouse model of mild trauma-related acute kidney injury after thorax trauma and hemorrhagic shock. In summary, we demonstrated that cryo-CECT is an effective 3D histopathological tool that allows to enhance our understanding of kidney tissue microstructure and their related function.

Abstract Cochlear implant restores hearing loss through electrical stimulation of the hearing nerve from within the cochlea. Unfortunately, surgical implantation of this neuroprosthesis often traumatizes delicate intracochlear structures, resulting in loss of residual hearing and compromising hearing in noisy environments and appreciation of music. To avoid cochlear trauma, insertion techniques and devices have to be adjusted to the cochlear microanatomy. However, existing techniques were unable to achieve a representative visualization of the human cochlea: classical histology damages the tissues and lacks 3D perspective; standard microCT fails to resolve the cochlear soft tissues; and previously used X-ray contrast-enhancing staining agents are destructive. In this study, we overcame these limitations by performing contrast-enhanced microCT imaging (CECT) with a novel polyoxometalate staining agent Hf-WD POM. With Hf-WD POM-based CECT, we achieved nondestructive, high-resolution, simultaneous, 3D visualization of the mineralized and soft microstructures in fresh-frozen human cochleae. This enabled quantitative analysis of the true intracochlear dimensions and led to anatomical discoveries, concerning surgically-relevant microstructures: the round window membrane, the Rosenthal’s canal and the secondary spiral lamina. Furthermore, we demonstrated that Hf-WD POM-based CECT enables quantitative assessment of these structures as well as their trauma.

The authors present cryogenic contrast-enhanced MicroCT (cryo-CECT), which by freezing stained samples at optimal freezing rates improves the visualization of the tissue microstructure. They demonstrate quantitative 3D analysis of individual tissue constituents, such as muscle and collagen fibers.

To improve our understanding of the brain microstructure, high-resolution 3D imaging is used to complement classical 2D histological assessment techniques. X-ray computed tomography allows high-resolution 3D imaging, but requires methods for enhancing contrast of soft tissues. Applying contrast-enhancing staining agents (CESAs) ameliorates the X-ray attenuating properties of soft tissue constituents and is referred to as contrast-enhanced computed tomography (CECT). Despite the large number of chemical compounds that have successfully been applied as CESAs for imaging brain, they are often toxic for the researcher, destructive for the tissue and without proper characterization of affinity mechanisms. We evaluated two sets of chemically related CESAs (organic, iodinated: Hexabrix and CA4+ and inorganic polyoxometalates: 1:2 hafnium-substituted Wells-Dawson phosphotungstate and Preyssler anion), for CECT imaging of healthy murine hemispheres. We then selected the CESA (Hexabrix) that provided the highest contrast between gray and white matter and applied it to a cuprizone-induced demyelination model. Differences in the penetration rate, effect on tissue integrity and affinity for tissue constituents have been observed for the evaluated CESAs. Cuprizone-induced demyelination could be visualized and quantified after Hexabrix staining. Four new non-toxic and non-destructive CESAs to the field of brain CECT imaging were introduced. The added value of CECT was shown by successfully applying it to a cuprizone-induced demyelination model. This research will prove to be crucial for further development of CESAs for ex vivo brain CECT and 3D histopathology.

Objective: The use of thiazolidinediones (TZDs) as insulin sensitizers has been shown to have side effects including increased accumulation of bone marrow adipocytes (BMAds) associated with a higher fracture risk and bone loss. A novel TZD analog MSDC-0602K with low affinity to PPARγ has been developed to reduce adverse effects of TZD therapy. However, the effect of MSDC-0602K on bone phenotype and bone marrow mesenchymal stem cells (BM-MSCs) in relation to obesity has not been intensively studied yet. Methods: Here, we investigated whether 8-week treatment with MSDC-0602K has a less detrimental effect on bone loss and BM-MSC properties in obese mice in comparison to first generation of TZDs, pioglitazone. Bone parameters (bone microstructure, bone marrow adiposity, bone strength) were examined by μCT and 3-point bending test. Primary BM-MSCs were isolated and measured for osteoblast and adipocyte differentiation. Cellular senescence, bioenergetic profiling, nutrient consumption and insulin signaling were also determined. Results: The findings demonstrate that MSDC-0602K improved bone parameters along with increased proportion of smaller BMAds in tibia of obese mice when compared to pioglitazone. Further, primary BM-MSCs isolated from treated mice and human BM-MSCs revealed decreased adipocyte and higher osteoblast differentiation accompanied with less inflammatory and senescent phenotype induced by MSDC-0602K vs. pioglitazone. These changes were further reflected by increased glycolytic activity differently affecting glutamine and glucose cellular metabolism in MSDC-0602K-treated cells compared to pioglitazone, associated with higher osteogenesis. Conclusion: Our study provides novel insights into the action of MSDC-0602K in obese mice, characterized by the absence of detrimental effects on bone quality and BM-MSC metabolism when compared to classical TZDs and thus suggesting a potential therapeutical use of MSDC-0602K in both metabolic and bone diseases.