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.     

MRT Letter: 3D culture of isolated cells: A fast and efficient method for optimizing their histochemical and immunocytochemical analyses

The rapid development of three‐dimensional (3D) culture systems and engineered cell‐based tissue models gave rise to an increasing need of new techniques, allowing the microscopic observation of cell behavior/morphology in tissue‐like structures, as clearly signalled by several authors during the last decennium. With samples consisting of small aggregates of isolated cells grown in suspension, it is often difficult to produce an optimal embedded preparation that can be further successfully processed for classical histochemical investigations. In this work, we describe a new, easy to use, efficient method that enables to embed an enriched “preparation” of isolated cells/small 3D cell aggregates, without any cell stress or damage. As for after tissue‐embedding procedures, the cellular blocks can be further suitably processed for efficient histochemical as well as immunohistochemical analyses, rendering more informative‐and attractive‐studies onto 3D cell‐based culture of neo‐tissues. Microsc. Res. Tech. 78:249–254, 2015. © 2015 Wiley Periodicals, Inc.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass‐based surfaces

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass‐based materials are due to altered mRNA and protein levels. Primary rat‐derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real‐time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals. Microsc. Res. Tech. 78:1046–1053, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

Huge clusters of embryonic stem cells in human embryos: A morphologic study

Background: Nothing is known about huge clusters (HC) of embryonic stem cells (ESC) in human fetal organs (HFO). Aim: To know the status of HC‐ESC in HFO. Methods: Morphology and immunohistochemistry (IHC) in 32 HFO of 7–40 gestational weeks (GW). Results: HC‐ESC were seen in many HFO including central nervous system, spinal cords, spine, soft tissue, bone, skin, thyroid, lung, liver, pancreas, gall bladder, extrahepatic bile duct, adrenal, kidney, bladder, foregut, midgut, hindgut, female and male genital organs, and neurons. HC‐ESC's were composed of two populations depending on constituting cells. One were large cells with ample acidophilic cytoplasms with vesicular nuclei and nucleoli. The other were small cells with scant cytoplasm with hyperchromatic nuclei without nucleoli, resembling lymphocytes. The HC‐ESC were frequently showed neuronal differentiation. HC‐ESC were positive for NCAM, synaptophysin, NSE, chromogranin, PDGFRA, AFP, ErbB2, bcl‐2, KIT, MET. They were negative for CD45, CD3, CD20, EMA, CEA, CA19‐9, cytokeratin (CK) 7, CK8, CK18, CK19, MUC1, MUC2, MUC5AC, and MUC6. The mean Ki‐67 labeling index (LI) was 13% ± 7%. HC‐ESC showed a little glycogen but lacked mucins. These HC‐ESC were seen in 7–25 GW, and they were rarely seen in 26–40 GW. Conclusions: The morphology, IHC, and ontogeny of HC‐ESC were described. Microsc. Res. Tech. 77:825–831, 2014. © 2014 Wiley Periodicals, Inc.     

In vivo second‐harmonic generation and ex vivo coherent anti‐stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb‐fiber laser‐based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 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.     

Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions

This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three‐dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro‐ and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast‐like or star‐like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. Microsc. Res. Tech. 78:792–800, 2015. © 2015 Wiley Periodicals, Inc.     

Synchrotron radiation X‐ray micro‐fluorescence: Protocol to study mesenchymal stem cells

The micro‐X‐ray fluorescence by synchrotron radiation (μ‐XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ‐XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10⁴ to 1 × 10⁷ were divided in two groups: G1: fixed in 4% paraformaldehyde diluted in phosphate‐buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10⁷ cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ‐XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10⁷ cells. Microsc. Res. Tech. 79:149–154, 2016. © 2016 Wiley Periodicals, Inc.     

A comparative study on the self‐assembly of an amyloid‐like peptide at water–solid interfaces and in bulk solutions

In the past years the self‐assembly of amyloid‐like peptides has attracted increasing attentions, because it is highly related to neurodegenerative diseases and has a potential for serving as nanomaterial to fabricate novel and useful nanostructures. In this paper, we focused on the role of interfacial conditions in the self‐assembly of an amyloid‐like peptide, termed Pep11. It was found that, when dissolved in bulk solutions, Pep11 formed into β‐sheet structures and assembled into long filaments in several hours, as revealed by Thioflavin T fluorescence and transmission electron microscopy (TEM) morphology characterization, respectively. When the peptide solution was added onto a mica/HOPG substrate, peptide filaments with three preferred orientations with an angle of 60° to each other were formed immediately, as imaged in situ by atomic force microscopy (AFM). However, the kinetics in filament formation and the morphologies of the formed beta sheet either on HOPG and mica or in bulk solutions were quite different. These results indicate that the interfacial properties dramatically affect the peptide self‐assembly process. Microsc. Res. Tech. 78:375–381, 2015. © 2015 Wiley Periodicals, Inc.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural analysis of murine hippocampal neural progenitor cells in culture

Despite the great number of studies devoted to neural stem/progenitor cell biology, the ultrastructural characteristics of these cells in vitro have not been fully studied. To determine the fine structure of hippocampal neural progenitor cells (NPCs) in culture, mouse fetal hippocampi (E18) were extracted, dissected, and cells were expanded as adherent monolayer culture. Electron microscopy revealed that NPCs had an immature phenotype, with a high nuclear/cytoplasmic ratio, small and scant organelles, underdeveloped endoplasmic reticulum, and many free ribosomes and polysomes. Our results may contribute to a better understanding of the fine structure and physiology of hippocampal NPCs in vitro. Microsc. Res. Tech. 78:128–133, 2015. © 2014 Wiley Periodicals, Inc.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 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.     

Real‐time in vivo confocal laser scanning microscopy of melanin‐containing cells: A promising diagnostic intervention

The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non‐invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin‐containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin‐containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin‐containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin‐containing cells and facilitate the clinical application of melanin‐containing cells in the investigation of dermatological disease. In summary, melanin‐containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin‐containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness. Microsc. Res. Tech. 78:1121–1127, 2015. © 2015 Wiley Periodicals, Inc.     

Human fetal ductal plate revisited. I. ductal plate expresses NCAM,KIT, MET,PDGFRA, and neuroendocrine antigens (NSE,chromogranin, synaptophysin,and CD56)

Background: The molecular mechanisms of ductal plate (DP) development and differentiation (DD) in human fetal livers (HFLs) are unclear. Materials and Methods: The author immunohistochemically investigated expressions of NCAM, KIT, KIT, PDGFRA, and neuroendocrine antigens in 32 HFLs. Results: The processes of human intrahepatic bile duct (IBD) DD could be categorized into four stages: DP, remodeling DP, remodeled DP, and mature IBD. NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. The biliary elements were positive for cytokeratin (CK)7, 8, 18, and 19. The hepatoblasts were positive for CK8 and CD18, but negative for CK7 and CK19; however, periportal hepatoblasts showed biliary‐type CKs (CK7 and CK19). NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. KIT was occasionally (12/32 cases) expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. NCAM expression was also seen in some hepatoblasts and hematopoietic cells and neurons. KIT was also expressed in some hepatoblasts, hematopoietic cells, and mast cells. MET and PDGFRA were strongly expressed in DP, remodeling DP, remodeled DP, and mature IBD. MET and PDGFRA were also strongly expressed in hepatoblasts and hematopoietic cells. MET and PDGFRA were not expressed in portal mesenchyme, portal veins, sinusoids, and hepatic veins. DP showed immunoreactive chromogranin, synaptophysin, neuron‐specific enolase (NSE), and CD56. Expressions of chromogranin and CD56 were infrequently seen in remodeling DP. No expressions of these four neuroendocrine antigens were seen in remodeled DP and mature IBD. The nerve fibers were consistently positive for chromogranin, synaptophysin, NSE, and CD56 in the portal mesenchyme in the stages of remodeling DP, remodeled DP, and mature IBDs. Conclusions: The data suggest that NCAM, KIT/stem cell factor‐signaling, NSE, hepatocyte growth factor/MET signaling, PDGFα/PDGFRA signaling, chromogranin, synaptophysin, and CD56 play important roles in DD of biliary cells of HFL. They also suggest that the DP cells having neuroendocrine molecules give rise to hepatic stem/progenitor cells. Microsc. Res. Tech. 77:814–824, 2014. © 2014 Wiley Periodicals, Inc.     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

Quantitative evaluation of bone resorption activity of osteoclast‐like cells by measuring calcium phosphate resorbing area using incubator‐facilitated and video‐enhanced microscopy

Quantitative evaluation of the ability of bone resorption activity in live osteoclast‐like cells (OCLs) has not yet been reported on. In this study, we observed the sequential morphological change of OCLs and measured the resorbing calcium phosphate (CP) area made by OCLs alone and with the addition of elcatonin utilizing incubator facilitated video‐enhanced microscopy. OCLs, which were obtained from a coculture of ddy‐mouse osteoblastic cells and bone marrow cells, were cultured on CP‐coated quartz cover slips. The CP‐free area increased constantly in the OCLs alone, whereas it did not increase after the addition of elcatonin. This study showed that analysis of the resorbed areas under the OCL body using this method enables the sequential quantitative evaluation of the bone resorption activity and the effect of several therapeutic agents on bone resorption in vitro. Microsc. Res. Tech, 2009. © 2008 Wiley‐Liss, Inc.