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(a) FE SEM 700x, CHO incus bone resorption bay at higher magnification, osteoclast snake trail pathway is visible (arrows). At the center of the resorption bay, a small promontory rises being relatively resistant to resorption. (b) FE SEM, 600x, osteoclastic resorption bay on osteoporotic human femur neck (arrows), they are unequivocally of osteoclast origin and are undistinguishable from those in (a).
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Bone erosion is considered a typical characteristic of advanced or complicated cholesteatoma (CHO), although it is still a matter of debate if bone erosion is due to osteoclast action, being the specific literature controversial. The purpose of this study was to apply a novel scanning characterization approach, the BSE 3D image analysis, to study t...
Citations
... Macrophages were grown on glass disks and processed as reported in [24][25][26][27], as follows: fixation fluid was glutaraldehyde 2.5% in PBS pH 7.4 at 4 • C for at least 48 h; washing solution used was PBS pH 7.4 20 min × 2 times; post-fixation solution was OsO 4 1.33% in H 2 O 1 h. Following washing in PBS pH 7.4 20 min × 2 times, dehydration was performed in ascending ethanol series (30%, 50%, 70%, 96% 10 min each, 100% 10 min × 3). ...
Since the discovery of graphene, there has been a wide range of the literature dealing with its versatile structure and easy binding of biomolecules as well as its large loading capacity. In the emerging field of immunotherapy, graphene and its derivatives have potential uses as drug delivery platforms directly into tumour sites or as adjuvants in cancer vaccines, as they are internalized by monocytes which in turn may activate adaptive anti-tumoral immune responses. In this study, we expose cells of the innate immune system and a human acute monocytic leukemia cell line (THP-1) to low doses of small-sized GO nanosheets functionalized with bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC), to study their acute response after internalization. We show by flow cytometry, uptake in cells of GO-BSA-FITC reaches 80% and cell viability and ROS production are both unaffected by exposure to nanoparticles. On the contrary, GO-BSA nanosheets seem to have an inhibitory effect on ROS production, probably due to their antioxidant properties. We also provided results on chemotaxis of macrophages derived from peripheral blood monocytes treated with GO-BSA. In conclusion, we showed the size of nanosheets, the concentration used and the degree of functionalization were important factors for biocompatibility of GO in immune cells. Its low cytotoxicity and high adaptability to the cells of the innate immune system make it a good candidate for deployment in immunotherapy, in particular for delivering protein antigens to monocytes which activate adaptive immunity.
... Previous studies confirmed that the resorptive process in cholesteatoma is driven by activated osteoclasts. In a scanning electron microscopy study on the morphometry of erosions in auditory ossicles affected by cholesteatoma, osteoclastic resorptive lacunae appeared similar to those in osteoporotic femoral necks [29]. Furthermore, osteoclasts have been shown to be activated by paracrine secretion of RANKL by fibroblasts and lymphocytes located in the peri-matrix and triggered by inflammation [9,30,31]. ...
Cholesteatoma can lead to progressive destruction of the auditory ossicles along with conductive hearing loss but precise data on the microstructural, cellular, and compositional aspects of affected ossicles are not available. Here, we obtained incus specimens from patients who had cholesteatoma with conductive hearing loss. Incudes were evaluated by micro-computed tomography, histomorphometry on undecalcified sections, quantitative backscattered electron imaging, and nanoindentation. Results were compared with two control groups taken from patients with chronic otitis media as well as from skeletally intact donors at autopsy. The porosity of incus specimens was higher in cholesteatoma than in chronic otitis media, along with a higher osteoclast surface per bone surface. Histomorphometric assessment revealed higher osteoid levels and osteocyte numbers in cholesteatoma incudes. Incudes affected by cholesteatoma also showed lower matrix mineralization compared with specimens from healthy controls and chronic otitis media. Furthermore, the modulus-to-hardness ratio was higher in cholesteatoma specimens compared with controls. Taken together, we demonstrated increased porosity along with increased osteoclast indices, impaired matrix mineralization, and altered biomechanical properties as distinct features of the incus in cholesteatoma. Based on our findings, a possible impact of impaired bone quality on conductive hearing loss should be further explored.
... of Supplementary Materials. Statistical analysis of data was performed by MedCalc© software (version 20.218, MedCalc Software ltd, Ostend, Belgium) and SPSS© statistical software (version 29, IBM©-SPSS© Statistics, Milan, Italy)[36,37]. ...
Glioblastoma is an aggressive brain tumor with an average life expectancy between 14 and 16 months after diagnosis. The Ki-67 labeling index (LI), a measure of cellular proliferation, is emerging as a prognostic marker in GBM. In this study, we investigated the ultrastructure of glioblastoma tissue from 9 patients with the same molecular profile (adult IDH wild-type glioblastoma, wild-type ATRX, and positive for TP53 expression, GFAP expression, and EGFR overexpression) to find possible ultrastructural features to be used as biomarkers and correlated with the only parameter that differs among our samples, the Ki-67 LI. Our main results were the visualization of the anatomical basis of astrocyte-endothelial cells crosstalk; the ultrastructural in situ imaging of clusters of hyperactivated microglia cells (MsEVs); the ultrastructural in situ imaging of microglia cells storing lipid vesicles (MsLVs); the ultrastructural in situ imaging of neoplastic cells mitophagy (NCsM). The statistical analysis of our data indicated that MsEVs and MsLVs correlate with the Ki-67 LI value. We can thus assume they are good candidates to be considered morphological biomarkers correlating to Ki-67 LI. The role of NCsM instead must be further evaluated. Our study findings demonstrate that by combining ultrastructural characteristics with molecular information, we can discover biomarkers that have the potential to enhance diagnostic precision, aid in treatment decision-making, identify targets for therapy, and enable personalized treatment plans tailored to each patient. However, further research with larger sample sizes is needed to validate these findings and fully utilize the potential of ultrastructural analysis in managing glioblastoma.
... Crystals surface measurements and 3D reconstructions were carried out on pictures captured at 2000× and 5000× magnification with the aid of Hitachi 3d Map software (Digital Surf, Beçason, France) [18]. Data were analyzed by MedCalc© statistical software Version 22.003 (MedCalc©, Ostende, Belgium). ...
Background: The use of effective, low-cost, and easy-to-use products for early caries management will avoid loss of dental vitality and impairment in oral function. The ability of fluoride to re-mineralize dental surfaces has been widely reported as well as vitamin D demonstrated to have significant potential in improving the remineralization of early lesions on enamel surfaces. The aim of the present ex vivo study was to evaluate the effect of a fluoride and vitamin D solution in terms of formation of mineral crystals on the enamel of primary teeth, and their permanence over time on dental surfaces. Methods: Sixteen extracted deciduous teeth were cut to obtain 64 specimens that were divided into two groups. The first consisted of immersion of specimens for 4 days in a fluoride solution (T1); in the second group, the specimens were immersed for 4 days (T1) in fluoride and Vitamin D solution, and for a further 2 (T2) and 4 days (T3) in saline solution. Then, samples were morphologically analyzed by using Variable Pressure Scanning Electron Microscope (VPSEM) and underwent 3D surface reconstruction. Results: After a 4-day immersion in both solutions, octahedral-shaped crystals were formed on the enamel surface of primary teeth, demonstrating any statistically significant differences in terms of number, size, and shape. Moreover, the binding of the same crystals seemed to be strong enough to be maintained until 4 days in saline solution. However, a partial dissolution was observed in a time-dependent manner. Conclusions: A topical application of fluoride and Vitamin D promoted the formation of persistent mineral crystals on enamel surfaces of deciduous teeth and should be further studied to be potentially used as an alternative strategy in preventive dentistry.
... Ultrathin (80e90 nm) sections were obtained using an ultramicrotome (Leica EM UC6, Vienna, Austria). For the TEM observation, the ultrathin sections were collected on 100-mesh copper grids (Assing, Rome, Italy) stained with a mix of lanthanides solution (Uranyless, Electron Microscopy Sciences) and lead citrate [90]. Imaging was performed using a transmission electron microscope (Carl Zeiss EM10, Thornwood, NY) with an accelerating voltage of 60 kV and a DEBEN XR80 AMT CCD camera. ...
Biocompatible gold nanoparticles (AuNPs) are particularly interesting for photo-thermal therapy (PTT) of cancer treatment because of their ability to convert light into heating efficiently. Nevertheless, the random accumulation of AuNPs in tissues, mainly determined by their retention time in the bloodstream, is one of the main limiting factors for their use in PTT applications. For this reason, efficient targeting and monitoring of AuNPs in the selected tissues is of paramount importance. This manuscript reports on a new generation of 99mTc-labeled AuNPs coated with keratin (Ker-AuNPs) and their spatial localization investigated by nuclear imaging techniques on an animal-free model. The effective radiolabeling of Ker-AuNPs with 99mTc is achieved using the chelating agent diethylenetriaminepentaacetic acid (DTPA), resulting in the 99mTc-DTPA-Ker-AuNPs nanoconjugate. The 99mTc-DTPA-Ker-AuNPs display a radiochemical purity of 90.7% and excellent photo-thermal properties. In addition, the biocompatibility of the 99mTc-DTPA-Ker-AuNPs with healthy human embryonic kidney (HEK293T) cells is shown. A Lab-On-a-Chip (LoC) approach is used to localize and study the stability of 99mTc-DTPA-Ker-AuNPs under dynamic conditions. To this end, the nanoconjugates are injected into a polydimethylsiloxane microfluidic chip mimicking the renal filtering unit, the nephron, and monitored via radio-imaging and thermo-optical experiments. These detailed studies establish that DTPA-assisted 99mTc-labeled Ker-AuNPs are excellent candidates as biocompatible and non-invasive radiolabeled nanotherapeutic for PTT-based applications.
... For each image, a selected area was extracted for the 3D image reconstruction procedure. The surface topography of the extracted area is shown in false colors [38], and it was processed by the particle count procedure to evaluate the size of ECM granules. ...
Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli, Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation.
... A single image reconstruction procedure was used, and a representative area was extracted from the 3D reconstructed image. The surface topography of the extracted area was shown in false colors [44] and used to extract data of the projected area of holes and peaks present on the extracellular matrix (ECM) surface in the control and treated samples. Data (from N = 50 extracted areas for each sample) were then statistically analyzed by the software MedCalc© (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; ...
Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.
... • [31] and Hitachi 3D Map (Digital Surf, Besancon, France) [32]. Data were statistically analyzed, and summary statistics, t-tests, and ANOVAs with Bonferroni correction were performed, and data were plotted in histograms. ...
Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes’ role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them “in situ” in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs’ exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.
... A previous histo-pathological study detected an increase in osteoclast density [81] and other studies have directly linked osteoclast activation to bone destruction found in MEC disease [81,184]. For osteoclast activation, the receptor activator for nuclear factor kappa B ligand (RANKL) has been identified as a key factor in promoting the differentiation and fusion of osteoclast precursor cells and activating the bone resorption by mature osteoclasts, while osteoprotegerin OPG on the other side is a decoy receptor for RANKL negatively regulating this process [185]. ...
Middle ear cholesteatoma (MEC), is a destructive, and locally invasive lesion in the middle ear driven by inflammation with an annual incidence of 10 per 100,000. Surgical extraction/excision remains the only treatment strategy available and recurrence is high (up to 40%), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review was targeted at connecting the dysregulated inflammatory network of MEC to pathogenesis and identification of pharmaceutical targets. We summarized the numerous basic research endeavors undertaken over the last 30+ years to identify the key targets in the dysregulated inflammatory pathways and judged the level of evidence for a given target if it was generated by in vitro, in vivo or clinical experiments. MEC pathogenesis was found to be connected to cytokines characteristic for Th1, Th17 and M1 cells. In addition, we found that the inflammation created damage associated molecular patterns (DAMPs), which further promoted inflammation. Similar positive feedback loops have already been described for other Th1/Th17 driven inflammatory diseases (arthritis, Crohn’s disease or multiple sclerosis). A wide-ranging search for molecular targeted therapies (MTT) led to the discovery of over a hundred clinically approved drugs already applied in precision medicine. Based on exclusion criteria designed to enable fast translation as well as efficacy, we condensed the numerous MTTs down to 13 top drugs. The review should serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history.
... A comparable honeycomb pattern is known in the tooth enamel and bones from continental aquatic environments (Fernández-Jalvo and Andrews, 2016). Howship's lacunae, which form during osteoclastic resorption, have a similar shape (Relucenti et al., 2020). Because the microstructures also have been observed on the periosteal surface of the bone, microorganisms such as cyanobacteria or algae appear to be the most likely causal agents. ...
Paleontological remains retrieved from permafrost represent the most informative records of Pleistocene ecosystems. Different levels of past microbial activity affecting fossil material preservation are presented for two selected bone samples—an almost intact Bison sp. metacarpus (45.0 ± 5.0 14C ka BP) and a weathered Equus sp. metacarpus (37.8 ± 1.7 14C ka BP) from the recently exposed cryogenic geo-contexts in the Yana River basin, NE Yakutia. Diagenetic changes in bone porosity and chemical composition as a result of the past microbial activity were investigated by multiple analytical methods. In the bison bone, which was permafrost-sealed shortly after death of the animal and conserved for ca. 45 ka in a frozen state in a cryolithic formation, only superficial microbial degradation processes were detected. Progressive microbial attacks characterize the
horse bone, which was exposed to MIS 3 sub-aerial biogenic decay and modern surficial weathering. This is evidenced by extensive bacterial micro-boring with the typical focal destructions, an increase in microbial porosity, and de-mineralized osseous zones due to waterlogged and poorly oxygenated past depositional conditions. New information contributes to better understanding of the diagenesis particularities and the associated chemical and biological agents of the fossil osteological assemblages with respect to their taphonomic and paleoenvironmental implications.
