Region‐specific localization of NOS isoforms and NADPH‐diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus)

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage‐dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)‐NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH‐d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH‐d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell‐specific localization in the efferent ductules and region‐ and cell‐specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech. 79:192–208, 2016. © 2016 Wiley Periodicals, Inc.     

FT‐Raman spectroscopy, µ‐EDXRF spectrometry,and microhardness analysis of the dentin of primary and permanent teeth

The chemical compositions (organic and inorganic contents) and mechanical behaviors of the dentin of permanent and deciduous teeth were analyzed and compared using X‐ray fluorescence spectrometry (µ‐EDXRF) Fourier transform Raman spectroscopy (FT‐Raman) and a microhardness test (HD). Healthy fresh human primary and permanent molars (n = 10) were selected, The buccal surfaces facing upwards were stabilized in an acrylic plate, flattened, polished, and submitted to the µ‐EDXRF, FT‐Raman, and HD analysis. The results of the analysis were subjected to ANOVAs and Mann‐Whitney U/Student's t multiple comparisons tests. The data showed similar values for the dentin of the primary and permanent teeth in P content, organic content (amide I peak), inorganic content ( – 430 and 590), and microhardness, Nevertheless, Ca content and Ca/P weight ratio were higher, and the peak was lower in the dentin of the permanent teeth compared to primary teeth. It be concluded that despite permanent teeth showed more Ca element, both substrates showed similar behavior of chemical and physical properties.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

1, 2 di‐substituted idopyranose from Vitex negundo l. Protects against streptozotocin‐induced diabetes by inhibiting nuclear factor‐kappa B and inducible nitric oxide synthase expression

The aim of this study is to investigate the mechanism of an action of compound isolated from Vitex negundo in streptozotocin‐induced diabetic mice. Light microscopic examination of liver, kidney and pancreatic sections of streptozotocin‐induced diabetic mice showed changes like coarsening of acinar cells of endoplasmic reticulum, destruction of β‐cells, and alteration in their secretory function were observed in the pancreas. Changes like dilation of vein, unusual concentric arrangement of hepatocytes, and liver fibrosis were observed in the liver. Thickening of tubules and expansion of glomerulus were observed in kidneys. All these altered parameters were reversed close to normal condition upon treatment using idopyranose. The results show the antidiabetic potential of idopyranose. Interestingly, liver, kidney, and pancreatic sections of diabetic mice fed with the isolated 1, 2 di‐substituted idopyranose showed regeneration of hepatocytes, nephrocytes, as well as β‐cells and acinar region appeared normal with increased numbers of β‐cells. To understand the probable mechanism of action of 1, 2 di‐substituted idopyranose, we analyzed proinflammatory inducible nitric oxide synthase (iNOS) and nuclear factor‐kappa B (NF‐κB) expression by immunohistochemistry and the results showed an increased iNOS and NF‐κB levels in streptozotocin‐induced diabetic liver, kidney and pancreas. Such high iNOS and NF‐κB levels were inhibited in 1, 2 di‐substituted idopyranose treated mice. The results suggest that 1, 2 di‐substituted idopyranose helps in the protection of hepatocytes, nephrocytes and pancreatic β‐cells probably by its action against NF‐κB and iNOS mediated inflammation in streptozotocin‐induced diabetes. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Confocal scanning optical microscopy of a 3‐million‐year‐old Australopithecus afarensis femur

Portable confocal scanning optical microscopy (PCSOM) has been specifically developed for the noncontact and nondestructive imaging of early human fossil hard tissues, which here we describe and apply to a 3‐million‐year‐old femur from the celebrated Ethiopian skeleton, “Lucy,” referred to Australopithecus afarensis. We examine two bone tissue parameters that demonstrate the potential of this technology. First, subsurface reflection images from intact bone reveal bone cell spaces, the osteocyte lacunae, whose density is demonstrated to scale negatively with body size, reflecting aspects of metabolism and organismal life history. Second, images of a naturally fractured cross section near to Lucy's femoral mid‐shaft, which match in sign those of transmitted circularly polarized light, reveal relative collagen fiber orientation patterns that are an important indicator of femoral biomechanical efficacy. Preliminary results indicate that Lucy was characterized by metabolic constraints typical for a primate her body size and that in her femur she was adapted to habitual bipedalism. Limitations imposed by the transport and invasive histology of unique or rare fossils motivated development of the PCSOM so that specimens may be examined wherever and whenever nondestructive imaging is required. SCANNING 31: 1–10, 2009. © 2009 Wiley Periodicals, Inc.     

Micromorphology,pharmacognosy, and bio‐elemental analysis of an important medicinal herb: Verbascum thapsus L.

The present study was conducted on characterization of morpho‐anatomical, phytochemical, and bio‐elemental analysis of root, stem, and leaf of Verbascum thapsus. Morphologically Verbascum is a biennial plant that flowers for a month and a half in mid‐ to late summer. Various organoleptic features of root, leaf, and stem were recorded. Anatomically the T. S of the root, stem, and leaf showed a typical dicot histological differentiation. Leaf possessed anomocytic stomata, crescent shape vascular bundles, and covered with long and stellate type trichomes while, stem contained collateral type of vascular bundles and a well‐developed pith to store phytochemicals responsible for various pharmacological activities. The powder drug study through scanning electron microscopy revealed the presence of various types of tissues. Branched, tree like and stellate trichomes in root and leaf help in absorption and reduce loss of water. These anatomical features are responsible for the survival of the plant as biennial. Four macro elements (Na, K, Ca, and Mg) and seven microelements (Cr, Cu, Fe, Mn, Ni, Zn, and Cd) and their concentrations in ppm were also studied using Atomic Absorption Spectroscopy. Phytochemical screening of methanolic extract showed existence of various secondary metabolites, while mucilage and anthraquinones was not detected. The present study helps to understand the taxonomic identification of the plant based on morpho‐anatomical features and throws the attention of the researchers to carry out the work for developing its various formulations, which can ultimately be beneficial for the human beings as well as animals.     

17‐beta‐estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines

Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2‐Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2‐methoxyestradiol analog, (8R, 13S, 14S, 17S)‐2‐Ethyl‐13‐methyl‐7, 8, 9, 11, 12,13, 14, 15, 16, 17‐decahydro‐6H‐cyclopenta[a]phenanthrane‐3, 17‐diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor‐positive breast adenocarcinoma cells line (MCF‐7), estrogen receptor‐negative highly metastatic breast cell line (MDA‐MB‐231) and a non‐tumorigenic breast epithelial cell line (MCF‐12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization‐optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS‐exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non‐tumorigenic breast cell lines. Findings warrant in‐depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech. 77:236–242, 2014. © 2014 Wiley Periodicals, Inc.     

Immune system involvement in the regulation of ovarian function and augmentation of cancer

Increasing evidence indicates a role for the immune system and mesenchymal-epithelial interactions in the regulation of ovarian function. Cytokines produced by mesenchymal cells can stimulate development and regression of ovarian structures. We report here that mesenchymal cells releasing surface molecules among epithelial cells--namely vascular pericytes and monocyte-derived cells (MDC)--and intraepithelial T lymphocytes are associated with oogenesis and formation of new primary follicles in both fetal and adult human ovaries. These activated mesenchymal cells interact with the ovarian surface epithelium, which appears to be a source of secondary germ cells and granulosa cells. Activated pericytes and MDC are also associated with stimulation of thecal development during selection of growing secondary follicles from the cohort of primary follicles. However, survival of the dominant follicle during mid-follicular phase selection is associated with a lack of activity of mesenchymal cells and retardation of thecal development, since immature granulosa cells lacking aromatase are unable to resist high levels of thecal androgens. Once the selected follicle matures (late follicular phase), it shows enhanced activity of thecal mesenchymal cells and advanced thecal development. Corpus luteum (CL) development is accompanied by a high activity of vascular pericytes and MDC. In mature CL and CL of pregnancy, luteal MDC and pericytes show a stable (inactive) state. Regression of the CL is associated with regression of pericytes, transformation of MDC into dendritic cells, infiltration by T lymphocytes, and binding of immunoglobulin G to the luteal cells. The immunoglobulin M (IgM) binds to young but not mature luteal cells. In the CL of pregnancy, IgM binds to luteal vessels, but not to luteal cells. Regressing CL shows IgM binding to both luteal cells and vessels. In ovarian cancers, highly activated MDC and sometimes activated pericytes (poorly differentiated carcinomas) are present. IgM binding is similar to that seen in the CL of pregnancy. These data indicate that vascular pericytes, MDC, T cells, and immunoglobulins may play an important role in the regulation of ovarian physiology and contribute to the augmentation of ovarian cancer growth.     

Immunohistochemical characterization of TH13‐L2 spinal ganglia neurons in sheep (Ovis aries)

Spinal ganglia (SG) neurons are commonly classified according to various specific features. The most widespread classification based on morphological and ultrastructural features subdivides SG neurons into light and small dark neurons. Using immunohistochemical, histochemical and lectin methods, it is possible to further subdivide the small dark neurons into two subpopulations: peptidergic and nonpeptidergic neurons. The majority of studies on SG neurons were carried out on mice and rats; there are few or no studies on large mammals. In this study, some of the widely used neuronal markers, neurofilament 200 kDa (NF200), substance P (SP), calcitonin gene‐related peptide (CGRP) and isolectin B4 (IB4), were employed to characterize neuronal nitric oxide synthase (nNOS)‐immunoreactivity (‐IR) in sheep (Ovis aries) SG (Th13‐L2) neurons. The majority of the SG neurons were IB4‐labeled (79 ± 10%), followed by NF200‐ (45 ± 4%), NOS‐ (44 ± 10%), SP‐ (42 ± 5%) and CGRP‐IR (35 ± 7%) neurons. The triple staining experiments showed that a higher percentage (75 ± 16%) of NOS‐IR neurons bound both IB4 and CGRP, or both IB4 and SP (49 ± 6%). The IB4 marker showed an unexpected staining pattern; in fact, IB4‐labeled neurons largely colocalized with NF200, usually considered a marker of light SG neurons, and with CGRP and SP. For this reason, IB4 cannot be employed in sheep to differentiate between light and dark neurons, or between peptidergic and nonpeptidergic neurons. These results suggest the importance of being cautious when comparing data among different species. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Immunolocalization of estrogen and progesterone receptors in ewe mammary glands

Immunopresence of estrogen receptor α (ERα), β (ERβ) and progesterone receptor (PR) were examined in the ewe mammary gland from prepubertal stage to involution. Immunolocalization of ERα revealed a strong positive staining in nuclei of cells composing terminal ductular units (TDUs) in prepubertal ewes. A mild immunoreactivity was identified in early lactating gland. During late lactation immunoreactive product to ERα was observed in the cytoplasm of glandular epithelial cells in all alveoli. In mammary glands at involution ERα positivity was clearly nuclear, with weak to moderate cytoplasmic staining. Cytoplasmic strong immunostaining for ERβ was detected in cells of TDUs, whereas some stromal cells exhibited nuclear staining. A nuclear ERβ immunostaining was observed at early lactation, instead during late lactation, the positivity for ERβ showed only a moderate cytoplasmic distribution. At involution, ERβ positivity was very moderate and detected just in the cytoplasm of shrunken alveoli. Scattered nuclear staining of PR was observed just in mammary glands at early lactation. These results showed that in the mammary glands of sheep both estrogen receptor isoforms were displayed during lactation cycle and that PR appeared just at early lactation, reflecting their regulatory role in alveolar cells. Microsc. Res. Tech. 76:955–962, 2013. © 2013 Wiley Periodicals, Inc.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

Glucose‐dependent insulinotropic polypeptide–producing K cells in dexamethasone‐treated rats

Some studies indicate that diabetes mellitus exerts an influence on the gastrointestinal tract and its diffuse neuroendocrine system (DNES) in regard to cellular density and neuroendocrine content. Since there is no data about relationship between experimentally induced non–insulin‐dependent (type 2) diabetes mellitus (NIDDM) on the gut K cells, the aim of our study was to investigate immunohistochemical, stereological and ultrastructural changes of rat K cells after 12 days of dexamethasone treatment. Twenty male Wistar rats aged 30 days were given daily intraperitoneally 2 mg kg^–1 dexamethasone (group DEX, 10 rats) or saline (group C, 10 rats) for 12 days. Tissue specimens were obtained from each antrum with corpus and different parts of the small (SI) and large intestine (LI) of all animals. Immunohistochemistry was carried out using antisera against the GIP and insulin. Transmission electron microscopy was also used. Although, according to the literature data, rat K cells are present in the duodenum and jejunum and, to a lesser extent, in the ileum, in the present study we observed that those cells were abundant also in all parts of the LI. We observed generally that GIP‐producing K cells were augmented in all parts of SI and decreased in the LI of DEX rats. Insulin immunoreactivity (ir) coexpressed with GIP‐ir in K cells and was stronger in the SI of DEX rats as compared with C rats. We also found by electron microscopy that small intestinal K cells have features not only of GIP‐secreted but also of insulin‐secreted cells. We concluded that dexamethasone treatment caused proliferation of K cells in the rat SI, and simultaneously transformation of GIP‐producing K cells to insulin‐synthesizing cells.     

Depletion of enteroendocrine and mucus‐secreting cells is associated with colorectal carcinogenesis severity and impaired intestinal motility in rats

This study investigated the relationship between enteroendocrine and mucus‐secreting cells distribution, the severity of colonic mucosal injury and intestinal motility in experimental colorectal carcinogenesis. Using a standardized murine model of colorectal carcinogenesis, eight‐weeks‐old female Wistar rats weighting 147.30 ± 29.15g were randomized into two groups receiving a subcutaneous injection of 0.9% saline (control) or the chemical carcinogen 1,2‐dimethylhydrazine (DMH) at 20 mg/kg per week during 10 weeks. Aberrant crypt foci (ACF) were more frequent in DMH group compared to control group (P < 0.001). The number of enteroendocrine and mucus‐secreting cells, and intestinal motility was reduced in DMH animals (P < 0.05). The distribution of enteric neurons was similar in both groups. In DMH animals there was a direct correlation between colonic motility and distribution of enteroendocrine (R² = 0.68, P < 0.05) and mucus‐secreting cells (R² = 0.77, P < 0.05). Inverse correlation between the number of ACF, mucus‐secreting cells (R² = ?0.57, P < 0.05), and enteroendocrine cells (R² = ?0.74, P < 0.05) was also observed. There was inverse correlation between the severity of the mucosal lesion, the number of mucus‐secreting cells (R² = ?0.83, P < 0.05) and enteroendocrine cells (R² = ?0.96, P < 0.05). There was a direct correlation between nucleolar organizer regions (AgNOR) and ACF number (R² = 0.62; P < 0.01). Inverse correlation was also found between AgNOR, the number of mucus‐secreting cells (R² = ?0.76; P < 0.001), and enteroendocrine cells (R² = ?0.86; P < 0.001). Taken together, the results indicated that colonic malignant transformation is related to depletion of mucus‐secreting and enteroendocrine cells, which was a useful indicator of the evolutionary status of intestinal neoplasm, partially explaining the intestinal motility disorders in the early stages of colorectal carcinogenesis. Microsc. Res. Tech. 79:3–13, 2016. © 2015 Wiley Periodicals, Inc.     

Immunohistochemical detection and quantification of T cells in the small intestine of Isospora suis-infected piglets--influence of fixation technique and intestinal segment

Quantification of immunohistochemical results constitutes an important tool in the analysis of cells and tissue that is not readily replaced by other techniques. For reliable quantification, it is essential to consider factors such as tissue fixation and tissue sampling. We report a study on the model of the intestine of Isospora suis‐infected piglets, in which we addressed (1) whether the quantity of detectable T cells in the intestinal mucosa is the same in formalin‐, HOPE®‐, and cryo‐conserved material or whether the amounts of T cells at least correlate with one another; and (2) whether single jejunal segments differ in regard to the quantity of mucosal T cells and variability of lymphocyte infiltration. Quantification of T cells in histological sections of different parts of the jejunum of 15‐22 day old piglets infected with I. suis was performed using an anti‐CD3‐antibody and stereological point counting. Area fractions of T‐cell profiles per intestinal mucosa profile were higher in cryo‐conserved samples than in HOPE®‐ and formalin‐conserved material but no correlation between different fixations could be found. The proximal part of the jejunum contained fewer T cells compared with mid‐ and end‐jejunum. Coefficients of variation did not differ between the intestinal segments. For quantification of T cells in the gut mucosa of piglets infected with I. suis, the cryo‐conserved mid jejunum seems most suitable in cases when unbiased sampling of the complete intestine is not feasible. It is generally not possible to compare quantitative results of immunostaining in samples conserved by different methods. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Immobilization and stretching of 5′‐pyrene‐terminated DNA on carbon film deposited on electron microscope grid

The immobilization and stretching of randomly coiled DNA molecules on hydrophobic carbon film is a challenging microscopic technique, which possess various applications, especially for genome sequencing. In this report the pyrenyl nucleus is used as an anchor moiety to acquire higher affinity of double stranded DNA to the graphite surface. DNA and pyrene are joined through a linker composed of four aliphatic methylene groups. For the preparation of pyrene‐terminated DNA a multifunctional phosphoramidite monomer compound was designed. It contains pyrenylbutoxy group as an anchor moiety for π‐stacking attachment to the carbon film, 2‐cyanoethyloxy, and diisopropylamino as coupling groups for conjugation to activated oligonucleotide chain or DNA molecule. This monomer derivative was suitable for incorporation into automated solid‐phase DNA synthesis and was attached to the 5′ terminus of the DNA chain through a phosphodiester linkage. The successful immobilization and stretching of pyrene‐terminated DNA was demonstrated by conventional 100 kV transmission electron microscope. The microscopic analysis confirmed the stretched shape of the negatively charged nucleic acid pieces on the hydrophobic carbon film. Microsc. Res. Tech. 78:994–1000, 2015. © 2015 Wiley Periodicals, Inc.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Influence of the artificial saliva storage on 3‐D surface texture characteristics of contemporary dental nanocomposites

The aim of this study was to analyse the influence of the artificial saliva on a three‐dimensional (3‐D) surface texture of contemporary dental composites. The representatives of four composites types were tested: nanofilled (Filtek Ultimate Body, FUB), nanohybrid (Filtek Z550, FZ550), microfilled (Gradia Direct, GD) and microhybrid (Filtek Z250, FZ250). The specimens were polymerised and polished by the multistep protocol (SuperSnap, Shofu). Their surface was examined, before and after 3 weeks’ exposure to artificial saliva storage. The surface texture was analysed using the atomic force microscope (AFM). The obtained images were processed to calculate the areal autocorrelation function (AACF), anisotropy ratio S_tr (texture aspect ratio), and structure function (SF). The log–log plots of SF were used to calculate fractal properties, such as fractal dimension D, and pseudo‐topothesy K. The analysis showed changes in surface anisotropy ratio S_tr values, which became higher, whereas the S_q roughness (root‐mean‐square) reduced after the artificial saliva storage. All the samples exhibited bifractal structure before the saliva treatment, but only half of them remained bifractal afterwards (GD, FZ250), whereas the other half turned into a monofractal (FUB, FZ550). The cube‐count fractal dimension D_cc was found to be material‐ and treatment‐insensitive.     

The effect of different ratios of nano‐sized hydroxyapatite fillers on the micro‐tensile bond strength of an adhesive resin

The purpose of this study was to evaluate the micro‐tensile bond strength (μTBS) of dentin bonding agents containing different ratios of nano‐sized hydroxyapatite fillers (HA). X‐ray diffraction analysis was used for characterization, and scanning electron microscope (SEM) analysis was used to determine the HA particle size after that HA were mixed a bonding agents without filler. Dentin bonding agents were divided into four groups according to addition of different ratios of nano‐sized hydroxyapatite fillers as 2% HA, 5% HA, 7% HA, and no‐filler control group. The teeth (n = 32) were sectioned with a low‐speed diamond blade under water cooling to expose the mid‐coronal dentin. Following the bonding application, restorations were applied incrementally. Each tooth was cut on the x and y axis, and each specimen was fixed to a testing device and stressed until failure occurred. The debonded specimens were examined under 250× magnification without a coating layer at 2.00 kV using a SEM to determine failure patterns. μTBS data were analyzed using a anova and Tukey's post hoc test. The failure mode data were analyzed using the Chi‐Square test. The maximum mean value of μTBS was in the 7% HA group, while the minimum mean value of μTBS was observed in the control group. 7% HA group was statistically significant and higher than other groups while there were no significant differences between the control, 2% HA, and 5% HA groups. According to SEM analysis, fracture analysis revealed that the mixed fracture type was seen more often than the other fracture types. The particle size and amount of HA fillers added to the adhesive resin seem to affect the success of the bond strength to the dentin. Adding different ratio nano‐sized HA fillers to the adhesive resin contributed positively to the immediate μTBS values in the dentin.     

Developing of N‐(4‐methylpyrimidine‐2‐yl)methacrylamide Langmuir–Blodgett thin film chemical sensor via quartz crystal microbalance technique

In the present work, the characterization and gas sensing properties of newly synthesized N‐(4‐methylpyrimidine‐2‐yl)methacrylamide ( N‐MPMA ) monomer Langmuir–Blodgett (LB) thin films were investigated. The UV–visible spectroscopy, quartz crystal microbalance (QCM), and atomic force microscopy were utilized to characterize N‐MPMA LB thin films. The surface behavior of N‐MPMA monolayer was stable and allowed an effective transfer at a surface pressure of 14 mN/m. The mass change/unit area value of the N‐MPMA LB thin film deposited quartz crystal surfaces was investigated. The amount of N‐MPMA LB thin film deposited on the substrate for bilayer was calculated as 228.72 ng (86.31 ng/mm²) and 12.5 Hz frequency shift was observed for each layer of the N‐MPMA film. The kinetic responses of N‐MPMA LB film against chloroform, dichloromethane, benzene, and toluene were measured via QCM system at room temperature. N‐MPMA QCM sensor results displayed that chloroform has the largest frequency shifts compared with the other vapors used in the present work and these results can be illuminating in terms of physical properties of organic vapors.