‘Effects of novel root repair materials on attachment and morphological behaviour of periodontal ligament fibroblasts: Scanning electron microscopy observation’

The aim of this study was to evaluate the adhesion of periodontal ligament fibroblasts (PDLs) on newly proposed root repair materials [Biodentine, MM‐MTA, polymethylmethacrylate (PMMA) bone cement, and SDR], in comparison with contemporary root repair materials [IRM, Dyract compomer, ProRoot MTA (PMTA), and Vitrebond]. Five discs from each material were fabricated in sterile Teflon molds, and the specimens were aged and prewetted in cell culture media for 96 hours. Three material discs were used for scanning electron microscopy (SEM) for the assessment of the attachment, density, and morphological changes in the PDLs, while two samples were used for energy dispersive x‐ray spectroscopy (SEM‐EDX) to determine the elemental composition of the materials. Human PDLs were plated onto the materials at a density of 10,000/well, and incubated for 3 days. The SEM micrographs were taken at different magnifications (500× and 5000×). In the SEM, the cells were attached and well spread‐out on the surfaces of the Biodentine, PMTA, and Dyract compomer, while varied cell densities and morphological alterations were observed in the Vitrebond, IRM, MM‐MTA, SDR, and PMMA bone cement groups. The SEM‐EDX analysis revealed a maximum calcium percentage in the PMTA specimens, as well a maximum silicon percentage in the Dyract compomer specimens. This in vitro study demonstrated that the Biodentine and Dyract compomer supported PDL cell adhesion and spreading. The PMTA presented a favorable scaffold for better attachment of the PDL cell aggregates. Therefore, the calcium and silicon content of a material may enhance the PDL cell attachment.     

Age-dependent changes in the nervous and endocrine control of the thymus

The immune system, especially the thymus, undergoes age-related modifications leading to structural and functional changes in the lymphoid organs and immunocompetent cells. Nevertheless, the consequences of thymic involution in the peripheral pool of T-cells are still a matter of controversy. The control of the thymic function is very complex and involves intrathymic signals, the autonomic nervous system, and the endocrine system. Both thymocytes and thymic stromal cells express receptors for a wide range of hormones, as well as for neurotransmitters and neuropeptides, thus affecting thymocytes maturation. This review summarizes the age-dependent variations in the extrathymic components of the thymic microenvironment, i.e., vegetative nerves and hormones, and the possible effects of those changes in the immune function.     

Involution dependent changes in distribution and localization of bax,survivin, caspase‐3, and calpain‐1 in the rat endometrium

The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis‐related proteins (bax and survivin) and enzymes (caspase‐3 and calpain‐1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase‐3, calpain‐1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain‐1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase‐3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non‐apoptotic activity of bax, caspase‐3, calpain‐1, and survivin. Microsc. Res. Tech. 79:285–297, 2016. © 2016 Wiley Periodicals, Inc.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

Ultrastructural features of the myotendinous junction of the sternomastoid muscle in Wistar rats: From newborn to aging

The myotendinous junction (MTJ) is a major area for transmitting force from the skeletal muscle system and acts in joint position and stabilization. This study aimed to use transmission electron microscopy to describe the ultrastructural features of the MTJ of the sternomastoid muscle in Wistar rats from newborn to formation during adulthood and possible changes with aging. Ultrastructural features of the MTJ from the newborn group revealed pattern during development with interactions between muscle cells and extracellular matrix elements with thin folds in the sarcolemma and high cellular activity evidenced through numerous oval mitochondria groupings. The adult group had classical morphological features of the MTJ, with folds in the sarcolemma forming long projections called “finger‐like processes” and sarcoplasmic invaginations. Sarcomeres were aligned in series, showing mitochondria near the Z line in groupings between collagen fiber bundles. The old group had altered “finger‐like processes,” thickened in both levels of sarcoplasmic invaginations and in central connections with the lateral junctions. We conclude that the MTJ undergoes intense activity from newborn to its formation during adulthood. With increasing age, changes to the MTJ were observed in the shapes of the invaginations and “finger‐like processes” due to hypoactivity, potentially compromising force transmission and joint stability. Microsc. Res. Tech. 75:1292–1296, 2012. © 2012 Wiley Periodicals, Inc.     

The role of mass spectrometry in the analysis of vitamin D compounds

This review highlights the superseding role of mass spectrometry in the structural characterization and quantitation of vitamin D compounds in comparison to other analytical methods (e.g., UV, bioassays) that lack the sensitivity and specificity of mass spectrometry. After a short introduction to the biochemistry of vitamin D compounds, an overview of the current techniques to characterize and quantitate vitamin D compounds is given with emphasis on the contribution of mass spectrometry. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:72–86, 2013     

Effect of two medium chain triglycerides‐supplemented diets on synaptic morphology in the cerebellar cortex of late‐adult rats

Ketogenic diets (KDs) have shown beneficial effects in experimental models of neurodegeneration, designating aged individuals as possible recipients. However, few studies have investigated their consequences on aging brain. Here, late‐adult rats (19 months of age) were fed for 8 weeks with two medium chain triglycerides‐supplemented diets (MCT‐SDs) and the average area (S), numeric density (Nv_s), and surface density (S_v) of synapses, as well as the average volume (V), numeric density (Nv_m), and volume density (V_v) of synaptic mitochondria were evaluated in granule cell layer of the cerebellar cortex (GCL‐CCx) by computer‐assisted morphometric methods. MCT content was 10 or 20%. About 10%MCT‐SD induced the early appearance of senescent patterns (decreased Nv_s and Nv_m; increased V), whereas 20%MCT‐SD caused no changes. Recently, we have shown that both MCT‐SDs accelerate aging in the stratum moleculare of CA1 (SM CA1), but are “antiaging” in the outer molecular layer of dentate gyrus (OML DG). Since GCL‐CCx is more vulnerable to age than OML DG but less than SM CA1, present and previous results suggest that the effects of MCT‐SDs in the aging brain critically depend on neuronal vulnerability to age, besides MCT percentage. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

The changes of gap junctions between pituitary folliculo‐stellate cells during the postnatal development of zucker fatty and lean rats

We investigated the effect of leptin on the postnatal development of gap junctions between folliculo‐stellate cells by using Zucker fatty (fa/fa) rats that have defects of the functional leptin receptor. Male Zucker fatty rats (fa/fa) and male Zucker lean rats (+/+) were used at each of the following postnatal ages: 20, 30, 40, 50, 60, 70, 80, 90 days, and 1 year. On one of the aforementioned dates, the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions, and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicular profile. In Zucker lean male rats, the number of gap junctions remained relatively constant from days 50 to 90 (0.44 ± 0.02 to 0.49 ± 0.03), and was similar in 1 year old rats (0.47 ± 0.03). These data were statistically higher compared to Zucker fatty male rats. In Zucker fatty male rats, very few gap junctions were observed in 30‐day‐old rats (0.04 ± 0.01: mean ± SE). This disruption of gap junction formation persisted, and the number of gap junctions remained constant and showed a low level from days 40 to 90 (0.11 ± 0.02 to 0.17 ± 0.02); this finding was similar in 1‐year‐old rats (0.17 ± 0.02). These observations indicate that the effect of leptin over the gap junction formation within the anterior pituitary glands was directly mediated by interaction with the functional leptin receptor present on the folliculo‐stellate cells. Microsc. Res. Tech. 77:31–36, 2014. © 2013 Wiley Periodicals, Inc.     

天然气替代丙烷切割在液压支架中的推广使用

通过对天然气和丙烷在金属切割应用中的特点及成本进行分析、试验、比较,总结出增效天然气比丙烷混合气体更适用于板料切割,采用天然气代替丙烷切割是一种必然趋势。     

The administration of nonmetabolizable glucose analogues fails to suppress the development of glycogen autophagy in newborn rat hepatocytes

The effects of parenteral administration of glucose, 3‐methylglucose (3MG), or 2‐deoxyglucose (2DG) on the glycogen autophagy were studied in the newborn rat liver using electron microscopy and biochemical methods. The administration of glucose resulted in hyperglycemia and prevented the mobilization of hepatocytic glycogen. It also prevented the development of autophagic vacuoles in general and inhibited the glycogen‐degrading activity of acid α‐1,4‐glucosidase. The nonphosphorylated and not further metabolized glucose analog 3MG also produced hyperglycemia, but increased acid glucosidase. Pretreating the newborns with the β‐adrenergic blocker propranolol inhibited the effects of 3MG. The phosphorylated but not fully metabolized glucose analog 2DG produced similar effects. The administration of xylitol to the newborns already treated with 2DG, suppressed acid glucosidase. The results of this and our previous studies suggest that glucose must be metabolized beyond its phosphorylation step to inhibit acid glucosidase activity. Microsc. Res. Tech. 73:1009–1014, 2010. © 2010 Wiley‐Liss, Inc.     

Si3N4／GCr15摩擦副在过氧化氢介质中的摩擦磨损性能研究

在SST-ST销-盘式摩擦磨损试验机上考察了Si3N4陶瓷盘/GCr15钢球摩擦副在不同体积分数过氧化氢H2O2介质中的摩擦磨损性能,通过扫描电子显微镜(SEM)和X射线能谱仪(EDS)对Si3N4陶瓷盘磨损表面进行了分析.结果表明:在去离子水条件下,Si3N4陶瓷盘摩擦表面形成的金属粘着转移层使Si3N4陶瓷盘与GCr15钢球之间的摩擦转变为金属与金属之间的摩擦,摩擦因数逐渐增大;在体积分数30%,60%和90%过氧化氢介质中,Si3N4陶瓷盘的主要磨损机制是微裂纹导致的剥落.     

Age-related changes of NADPH-diaphorase-positive neurons in the rat inferior colliculus and auditory cortex

Nitric oxide (NO) has been implied in age-related changes of the central nervous system (CNS) and the central auditory pathway. The present study was conducted to investigate whether the number of NO-producing cells and their morphometric characteristics in the inferior colliculus (IC) and the auditory cortex (AC) are changed with the increasing age of the subjects. IC and AC sections of adult and senile Wistar rats were studied using the histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS). Our results showed a decreased area of the somas of NADPH-d-positive neurons in the dorsal cortex (DC) of the IC and a diffuse loss of NADPH-d-positive neurons in the senile IC and primary cortical auditory area (Te1). However, an increased number of NO-producing cells have been shown by other authors in different parts of the ageing auditory pathway and CNS. It seems that age-related changes in NADPH-d-positive cells may follow a region-specific route. These changes may be related to hearing impairments with increasing age.     

Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in man

Changes in the patterns of production and in the effects of signal substances may be involved in the development of tendinosis, a chronic condition of pain in human tendons. There is no previous information concerning the patterns of sympathetic innervation in the human patellar tendon. In this study, biopsies of normal and tendinosis patellar tendons were investigated with immunohistochemical methods, including the use of antibodies against tyrosine hydroxylase (TH) and neuropeptide Y, and against alpha1-, alpha2A-, and beta1-adrenoreceptors. It was noticed that most of the sympathetic innervation was detected in the walls of the blood vessels entering the tendon through the paratendinous tissue, and that the tendon tissue proper of the normal and tendinosis tendons was very scarcely innervated. Immunoreactions for adrenergic receptors were noticed in nerve fascicles containing both sensory and sympathetic nerve fibers. High levels of these receptors were also detected in the blood vessel walls; alpha1-adrenoreceptor immunoreactions being clearly more pronounced in the tendinosis tendons than in the tendons of controls. Interestingly, immunoreactions for adrenergic receptors and TH were noted for the tendon cells (tenocytes), especially in tendinosis tendons. The findings give a morphological correlate for the occurrence of sympathetically mediated effects in the patellar tendon and autocrine/paracrine catecholamine mechanisms for the tenocytes, particularly, in tendinosis. The observation of adrenergic receptors on tenocytes is interesting, as stimulation of these receptors can lead to cell proliferation, degeneration, and apoptosis, events which are all known to occur in tendinosis. Furthermore, the results imply that a possible source of catecholamine production might be the tenocytes themselves     

Morphology of the intralobular duct of the submandibular gland in rats in case of experimental diabetes mellitus

The objective of study is to study the peculiarities of morphological changes in different subdivisions of the intralobular duct of the submandibular gland (SMG) in rats in case of experimental diabetes mellitus (DM). The study included sexually mature male Wistar rats. Experimental DM was induced by streptozotocin. Electron microscopic study of subdivisions of the intralobular duct of the SMG was carried out on the 14th, 28th, 42nd, 56th, and 70th days of the experiment. In early stages of experimental DM the intercalated ducts are characterized by a relatively unchanged structure, and in late stages vacuolization of the cytoplasm of their epithelial cells is observed. Since the 14th day vacuolization of mitochondria is observed in epithelial cells of the granular ducts being the most pronounced on the 28th day and not apparent over the subsequent periods. The degree of filling with granules reduces till 56th day, however, it increases sligthly on the last day of the experiment. On the 28th–70th days vacuolization of the cytoplasm is observed in epithelial cells of the striated ducts. In addition, on the 14th day the mitochondrial matrix of these cells condenses; over the next periods it becomes enlightened and mitochondrial cristae are clearly visualized and disorganized. Conclusion: In the intralobular duct of the SMG in experimental DM dystrophic changes of different intensity occur in the granular and striated ducts on the 14th day and in the intercalated ducts only since the 42nd day of the experiment.     

Real‐time monitoring of the penetration of amphiphilic acrylate copolymer in leather using a fluorescent copolymer as tracer

A fluorescent tracer, poly (acrylic‐co‐stearyl acrylate‐co?3‐acryloyl fluorescein) [poly (AA‐co‐SA‐co‐Ac‐Flu)], used for real‐time monitoring the penetration of amphiphilic acrylate copolymer, poly (acrylic‐co‐stearyl acrylate) [poly (AA‐co‐SA)], in leather was synthesized by radical polymerization of acrylic, stearyl acrylate and fluorescent monomer, 3‐acryloyl fluorescein (Ac‐Flu). The structure, molecular weight, introduced fluorescent group content and fluorescent characteristics of the fluorescent tracer and target copolymer, amphiphilic acrylate copolymer, were also characterized. The results show that the tracer presents the similar structural characteristics to the target and enough fluorescence intensity with 1.68 wt % of the fluorescent monomer introduced amount. The vertical section of the leather treated with the target copolymer mixing with 7% of the tracer exhibits evident fluorescence, and the change of fluorescence intensity along with the vertical section with treating time increasing can reflect the penetration depth of the target copolymer. The introduction of the fluorescent group in polymer structure through copolymerization with a limited amount of fluorescent monomer, Ac‐Flu, is an effective way to make a tracer to monitor the penetration of the target in leather, which provides a new thought for the penetration research of syntans such as vinyl copolymer materials in leather manufacture. Microsc. Res. Tech. 78:1146–1153, 2015. © 2015 Wiley Periodicals, Inc.     

Autophagosomal glycogen‐degrading activity and its relationship to the general autophagic activity in newborn rat hepatocytes: The effects of parenteral glucose administration

The effects of the administration of parenteral glucose on the postnatal glycogen autophagic activity and its relationship to the general autophagic activity, were studied in newborn rat liver using electron microscopy and biochemical methods. Glucose abolished the normal postnatal hypoglycemia and preserved the hepatocytic hyaloplasmic glycogen to the levels of birth. It also inhibited the normal postnatal increase in the number and volume of autophagic vacuoles. Glucose especially decreased the rate of postnatal development of the glycogen‐containing autophagic vacuoles. This decrease was greater than that of the autophagic vacuoles in general. In the control animals at the age of 6 h, the total volume of the glycogen‐containing autophagic vacuoles accounted for 87% of the autophagic vacuoles in general, whereas in the glucose‐treated animals of the same age, for only 62%. The results of this and previous studies support the view that the general autophagic activity that develops in the immediate postnatal period in rat hepatocytes is mainly expressed as glycogen autophagic activity selectively inhibited by glucose. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).     

Potential limitations in the use of KillerRed for fluorescence microscopy

KillerRed, a bright red fluorescent protein, is a genetically encoded photosensitizer, which generates radicals and hydrogen peroxide upon green light illumination. The protein is a potentially powerful tool for selective light-induced protein inactivation and cell killing, and can also be used to study downstream effects of locally increased levels of reactive oxygen species. The initial aim of this study was to investigate whether or not KillerRed-mediated reactive oxygen species production inside peroxisomes could trigger the sequestration of these organelles into autophagosomes. Green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 was used as autophagosome marker. We observed that KillerRed also emits weak green fluorescence upon excitation at 480 nm, and this may lead to erroneous data interpretation in conditions where green fluorophores are used. We discuss this potential pitfall of KillerRed for biological imaging and formulate recommendations to avoid misinterpretation of the data.