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.

Objectives: The aim of this matched prospective cohort study was to examine the microarchitecture of the augmented bone following a modified alveolar ridge splitting procedure and compare it to that of native bone. Methods: In the test group, patients underwent a modified ridge split osteotomy procedure to restore the width of the posterior segment of the mandible. Patients with sufficient bone width for dental implant placement in the posterior region of the mandible following 3-month-long spontaneous healing after tooth removal were included in the control group. In both study groups, bone biopsy samples were harvested and dental implants were placed. Histomorphometry and micro-CT analysis were performed. Results: Altogether, 15 patients were included in this study (7 patients in the test group, with 14 bone core biopsies harvested, and 8 patients in the control group, with 13 bone core biopsies harvested). Percentage bone volume (BV/TV) in the micro-CT analysis (22.088 ± 8.094% and 12.075 ± 4.009% for the test and control group, respectively) showed statistically significant differences between study groups. Conclusions: Based on histological and micro-CT analyses, the modified ridge splitting procedure with autologous bone block harvested from the retromolar area results in a dental implant recipient bone microarchitecture superior to that of the extraction sockets left to heal undisturbed for a 3-month-long healing period.

Promising research results have been obtained on the tissue-regeneration properties of PRF (platelet-rich fibrin) in dentistry and maxillofacial surgery. PRF presumably promotes healing and accelerates ossification. In this case report, the patient had a history of Gorlin–Goltz syndrome, also called nevoid basal cell carcinoma syndrome, an autosomal dominant neurocutaneous disease that was known for many years. As a consequence, cysts were detected in both the mandible and maxilla. We performed decompression on this 37-year-old patient, followed by a cystectomy on an extensive lesion in the right angle of the mandible. One cyst from each side of the body mandible and one from the maxilla were completely enucleated, as determined using an intraoral exploration. The resulting bone defect was filled with a composite graft composed of a mixture of A-PRF and a serum albumin-coated bone allograft (BoneAlbumin). The wound was then covered with a PRF membrane. The surgical sites were closed per primam. The postoperative period was uneventful. Biopsies were performed after three and six months of healing for histological micromorphometry analyses. Dental implants were placed at the sampling site. Three months after the implantation, the ossified implants were fitted with superstructures. To date, no complications have appeared with the bone augmentation. The authors interpret from the findings that the combination of A-PRF and BoneAlbumin can be validated as a prosperous bone substitute. It can be safely implanted after a 3-month ossification period.

Background Src-like adaptor protein (SLAP), an adaptor between E3 ubiquitin ligase c-Cbl and the TCR complex, is a negative regulator of T-cell activation. SLAP regulates the expression of the CD3 ζ-chain. In accordance with our previous data, SLAP expression is increased in the CD4+ T-cells of patients with rheumatoid arthritis (RA). Recent data demonstrate that SLAP deficiency can prevent zymosan-induced arthritis in mice. Objectives We investigated the role of SLAP in collagen-induced arthritis (CIA). Methods Homozygous and heterozygous SLAP knock out and wild type (WT) C57BL/6J mice were used. After the isolation of genomial DNA, the genotype of the mice was determined. Then the animals were injected once (primary immunization) or twice (secondary immunization) with type II. collagen, or left untreated (the first injection was applied together with complete Freund’s adjuvant). Mice were monitored twice a week for 70 days, and the severity scores were evaluated. The scores were calculated for each limb (1 limb score: 0=no swelling; 4=the severe swelling). Results SLAP deficiency provided almost complete protection from the primary immunization of CIA. By contrast secondary immunization led to similar arthritis of the SLAP-KO, heterozygous or WT mice. Conclusion The authors’ present data suggest that SLAP deficiency protects dose-dependently from CIA. Acknowledgements This work was supported by OTKA/NKFIHO NN 111023; OTKA/NKFIHN K 131479. Disclosure of Interests None declared

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.

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.

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.

Background and objectives Osteoclasts are the unique bone-resorbing cells of hematopoietic origin; their development is regulated primarily by M-CSF, RANKL and integrin-mediated interactions. Our workgroup has previously tested thousands of Src-family kinase inhibitors produced by the Vichem Ltd on neutrophil granulocytes, which cells show many similarities with osteoclasts regarding signalling pathways. One of the most effective inhibitors was the PD166326, an anti-leukaemia drug candidate. Here we tested the effect of PD166326 on osteoclasts. Materials and methods Bone marrow cells were isolated from the long bones of C57Bl/6 mice on the 0. day and differentiated into osteoclasts in vitro in the presence of recombinant M-CSF and RANKL. Cultures were treated with PD166326 during different stages of osteoclastogenesis. Vehicle control samples received dimethyl sulfoxide (DMSO). Osteoclast differentiation was examined after 4 or 6 days by osteoclast-specific tartrate-resistant acid phosphatase (TRAP)-staining. Survival assay was performed at the 4th day using AnnexinV-PE and 7-AAD apoptosis and necrosis kit. Actin ring formation was observed with Lifeact EGFP transgenic mice. Results The number and the size of developing osteoclasts strongly decreased in case of the early administration of the inhibitor. The treatment of the cultures by 10 nM PD166326 practically blocked osteoclast development. When we administered the inhibitor later, from the 4th day – when the last main point of osteoclast development happens – only minor reduction was observed. The tendency to apoptosis did not increase in the PD166326 treated cultures. The administration of PD166326 did not influence the degradation of actin rings. Conclusions The anti-cancer drug PD166326 inhibited osteoclastogenesis in a very small concentration. We suppose that it has an effect on the early stage of osteoclast development. These data may be considered in the therapy of different cancers especially in metastatic bone diseases.

Vol. 66, No. 8, August 2014, pp 2210–2221 DOI 10.1002/art.38660 © 2014 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 PI3Kβ 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 PI3Kβ in osteoclast development and function using a combined genetic and pharmacologic approach. METHODS:The role of PI3Kβ in primary human and murine osteoclast cultures was tested with the PI3Kβ-selective inhibitor TGX221 and by using PI3Kβ(-/-) mice. The trabecular bone architecture of PI3Kβ(-/-) mice was evaluated using micro-computed tomography and histomorphometric analyses. RESULTS:The expression of PI3Kβ 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 PI3Kβ inhibitor TGX221 or by the genetic deficiency of PI3Kβ. This was likely due to defective cytoskeletal reorganization and vesicular trafficking, since PI3Kβ(-/-) 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. PI3Kβ(-/-) mice had significantly increased trabecular bone volume and showed abnormal osteoclast morphology with defective resorption pit formation. CONCLUSION:PI3Kβ plays an important role in osteoclast development and function and is required for in vivo bone homeostasis.