Are Artificial Intelligence-Assisted Three-Dimensional Histological Reconstructions Reliable for the Assessment of Trabecular Microarchitecture?

Objectives: This study aimed to create a three-dimensional histological reconstruction through the AI-assisted classification of tissues and the alignment of serial sections. The secondary aim was to evaluate if the novel technique for histological reconstruction accurately replicated the trabecular microarchitecture of bone. This was performed by conducting micromorphometric measurements on the reconstruction and comparing the results obtained with those of microCT reconstructions. Methods: A bone biopsy sample was harvested upon re-entry following sinus floor augmentation. Following microCT scanning and histological processing, a modified version of the U-Net architecture was trained to categorize tissues on the sections. Detector-free local feature matching with transformers was used to create the histological reconstruction. The micromorphometric parameters were calculated using Bruker's CTAn software (version 1.18.8.0, Bruker, Kontich, Belgium) for both histological and microCT datasets. Results: Correlation coefficients calculated between the micromorphometric parameters measured on the microCT and histological reconstruction suggest a strong linear relationship between the two with p-values of 0.777, 0.717, 0.705, 0.666, and 0.687 for BV/TV, BS/TV, Tb.Pf Tb.Th, and Tb.Sp, respectively. Bland-Altman and mountain plots suggest good agreement between BV/TV measurements on the two reconstruction methods. Conclusions: This novel method for three-dimensional histological reconstruction provides researchers with a tool that enables the assessment of accurate trabecular microarchitecture and histological information simultaneously.

A New Zebrafish Model for Pseudoxanthoma Elasticum.

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.

Hematopoietic or Osteoclast-Specific Deletion of Syk Leads to Increased Bone Mass in Experimental Mice.

Syk is a non-receptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk-/- mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (SykΔOC ) or hematopoietic (SykΔHaemo ) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both SykΔOC and SykΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Sykflox allele revealed complete and early deletion of Syk from SykΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from SykΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the SykΔOC and SykΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.

Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.

The phosphoinositide 3-kinase isoform PI3Kß regulates osteoclast-mediated bone resorption in humans and mice.

OBJECTIVE: While phosphoinositide 3-kinases (PI3Ks) are involved in various intracellular signal transduction processes, the specific functions of the different PI3K isoforms are poorly understood. We have previously shown that the PI3Kß isoform is required for arthritis development in the K/BxN serum-transfer model. Since osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis and other diseases, we undertook this study to test the role of PI3Kß in osteoclast development and function using a combined genetic and pharmacologic approach. METHODS: The role of PI3Kß in primary human and murine osteoclast cultures was tested with the PI3Kß-selective inhibitor TGX221 and by using PI3Kß(-/-) mice. The trabecular bone architecture of PI3Kß(-/-) mice was evaluated using micro-computed tomography and histomorphometric analyses. RESULTS: The expression of PI3Kß was strongly and specifically up-regulated during in vitro osteoclast differentiation. In vitro development of large multinucleated osteoclasts from human or murine progenitors and their resorption capacity were strongly reduced by the PI3Kß inhibitor TGX221 or by the genetic deficiency of PI3Kß. This was likely due to defective cytoskeletal reorganization and vesicular trafficking, since PI3Kß(-/-) mouse multinucleated cells failed to form actin rings and retained intracellular acidic vesicles and cathepsin K. In contrast, osteoclast-specific gene expression and the survival and apoptosis of osteoclasts were not affected. PI3Kß(-/-) mice had significantly increased trabecular bone volume and showed abnormal osteoclast morphology with defective resorption pit formation. CONCLUSION: PI3Kß plays an important role in osteoclast development and function and is required for in vivo bone homeostasis.