Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

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Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice

Zygote ◽

10.1017/s0967199421000733 ◽

2021 ◽

pp. 1-8

Author(s):

Mohammad Veisi ◽

Kamran Mansouri ◽

Vahideh Assadollahi ◽

Cyrus Jalili ◽

Afshin Pirnia ◽

...

Keyword(s):

Sertoli Cells ◽

Periodic Acid ◽

Three Dimensional ◽

3D Culture ◽

Alginate Hydrogel ◽

Periodic Acid Schiff ◽

Expression Levels ◽

Pas Staining

Summary An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.

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Control of Liver Tissue Reconstitution in Mesenteric Leaves: The Effect of Preculture on Mouse Hepatic Progenitor Cells Prior to Transplantation

Journal of Robotics and Mechatronics ◽

10.20965/jrm.2013.p0698 ◽

2013 ◽

Vol 25 (4) ◽

pp. 698-704 ◽

Cited By ~ 1

Author(s):

Nobuhiko Kojima ◽

◽

Yasuyuki Sakai

Keyword(s):

Progenitor Cells ◽

Periodic Acid ◽

Basal Medium ◽

Hepatic Progenitor Cells ◽

Periodic Acid Schiff ◽

3 Dimensional ◽

Enhancer Binding Protein ◽

Pas Staining ◽

Highly Porous

Our objective is to control the reconstitution of liverlike tissues at extrahepatic sites using hepatic progenitor cells (HPCs) andin vitropreculture prior to transplantation. We prepared cell-based hybrid grafts by culturing HPCs isolated from fetal E14.5 mouse livers on biodegradable, highly porous 3-dimensional poly-L-lactic acid (PLLA) scaffolds for 1 week in basal medium (the basal condition) or 10 mM nicotinamide (NA) and 1% dimethyl sulfoxide (DMSO) supplemented conditions (the ND-positive condition) prior to implantation. Sections of hybrid grafts cultured for 1 week showed that HPCs grew and spread on the surface of scaffolds under both basal and ND (+) conditions. Most of these cells were albumin (+) and CK18 (+). CK19 (+) cells were also present under the basal condition but not the ND (+) condition. Cultured hybrid grafts were implanted into the mesenteric leaves of mice and removed after 1 month. Transplanted tissues cultured under the basal condition consisted of albumin (+) hepatocyte-like and CK19 (+) biliary epithelial cell (BEC)-like cells organized in duct-like structures. In contrast, integrated tissues cultured under the ND (+) condition alone had differentiated albumin (+) hepatocyte-like cells and were relatively larger than those under the basal condition. Hepatocyte-like cells of transplanted hybrid grafts cultured under both conditions were periodic acid-Schiff (PAS) staining-positive and expressed transcription factors, hepatocyte nuclear factor (HNF) 4 and CCAAT/enhancer-binding protein (C/EBP) α. These findings suggest that combining progenitor cells andin vitropreculture may potentially regulate liverlike tissues at extrahepatic sites.

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Low doses of ethanol have Ca2+ ionophore-like effects on apical membrane potential of in vitro Necturus antrum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.261.1.g92 ◽

1991 ◽

Vol 261 (1) ◽

pp. G92-G103

Author(s):

M. J. Rutten ◽

C. D. Moore

Keyword(s):

Membrane Potential ◽

Apical Membrane ◽

Periodic Acid ◽

Ethanol Exposure ◽

Membrane Potentials ◽

Antral Mucosa ◽

Low Doses ◽

Periodic Acid Schiff ◽

Induced Changes

The effects of low doses of luminal ethanol on the amiloride-sensitive apical membrane potential of Necturus antral mucosa were studied using conventional microelectrode techniques. Luminal ethanol (0.250-4.0% vol/vol) caused a dose-dependent hyperpolarization of the apical membrane potential (Vmc), an increase in transepithelial resistance (Rt) and resistance ratio (Ra/Rb), and a decrease in transepithelial potential (Vms). Luminal amiloride (100 microM) to 4% ethanol-treated antra did not cause any additional hyperpolarization of Vmc. Compared with luminal 2% ethanol-Ringer, an equivalent osmotic mannitol solution depolarized Vmc and basolateral potential (Vcs), decreased Rt and Ra/Rb, and increased Vms. A single dose of 0.50% ethanol attenuated the effects of a second 2% ethanol exposure on Vmc. No change in periodic acid-Schiff (PAS)-positive mucous granule content could be found between control and 2% ethanol-treated antra. The Ca2+ ionophores A23187 or ionomycin (0.25-5.0 microM) dose dependently hyperpolarized the Vmc and Vcs, increased Rt and Ra/Rb, and decreased Vms. Luminal Ca(2+)-free Ringer had no effect on luminal 2.00% ethanol-induced changes in membrane potentials or resistances. Pretreatment with BAPTA blocked by approximately 70 and 55% the Vmc hyperpolarization of 2 and 4% ethanol, respectively. Pretreatment with ruthenium red (10-50 microM) also dose dependently reduced the 2% ethanol-induced changes in Vmc. The data indicate that 1) low doses of luminal ethanol and Ca2+ ionophores have similar effects on Necturus gastric antral membrane potentials and resistances, 2) ethanol-induced hyperpolarizations of the Vmc are partially mediated through an alteration in intracellular Ca2+, and 3) low doses of luminal ethanol do not cause the release of antral epithelial mucous granules at the time when significant changes are occurring in the Vmc.

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Necroptosis Contributes to Urban Particulate Matter-Induced Airway Epithelial Injury

Cellular Physiology and Biochemistry ◽

10.1159/000488726 ◽

2018 ◽

Vol 46 (2) ◽

pp. 699-712 ◽

Cited By ~ 13

Author(s):

Feng Xu ◽

Man Luo ◽

Lulu He ◽

Yuan Cao ◽

Wen Li ◽

...

Keyword(s):

Particulate Matter ◽

Pulmonary Inflammation ◽

Periodic Acid ◽

Enzyme Linked Immunosorbent Assay ◽

Periodic Acid Schiff ◽

Mucus Production ◽

Gm Csf ◽

Mucin Expression

Background/Aims: Necroptosis, a form of programmed necrosis, is involved in the pathologic process of several kinds of pulmonary diseases. However, the role of necroptosis in particulate matter (PM)–induced pulmonary injury remains unclear. The objective of this study is to investigate the involvement of necroptosis in the pathogenesis of PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction, both in vitro and in vivo. Methods: PM was administered into human bronchial epithelial (HBE) cells or mouse airways, and the inflammatory response and mucus production were assessed. The mRNA expressions of IL6, IL8 and MUC5AC in HBE cells and Cxcl1, Cxcl2, and Gm-csf in the lung tissues were detected by quantitative real-time RT-PCR. The secreted protein levels of IL6 and IL8 in culture supernatants and Cxcl1, Cxcl2, and Gm-csf in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). We used Western blot to measure the protein expressions of necroptosis-related proteins (RIPK1, RIPK3, and Phospho-MLKL), NF-κB (P65 and PP65), AP-1 (P-c-Jun and P-c-Fos) and MUC5AC. Cell necrosis and mitochondrial ROS were detected using flow cytometry. In addition, pathological changes and scoring of lung tissue samples were monitored using hemoxylin and eosin (H&E), periodic acid-schiff (PAS) and immunohistochemistry staining. Results: Our study showed that PM exposure induced RIP and MLKL-dependent necroptosis in HBE cells and in mouse lungs. Managing the necroptosis inhibitor Necrostatin-1 (Nec-1) and GSK’872, specific molecule inhibitors of necroptosis, markedly reduced PM-induced inflammatory cytokines, e.g., IL6 and IL8, and MUC5AC in HBE cells. Similarly, administering Nec-1 significantly reduced airway inflammation and mucus hyperproduction in PM-exposed mice. Mechanistically, we found PM–induced necroptosis was mediated by mitochondrial reactive oxygen species-dependent early growth response gene 1, which ultimately promoted inflammation and mucin expression through nuclear factor κB and activator protein-1 pathways, respectively. Conclusions: Our results demonstrate that necroptosis is involved in the pathogenesis of PM–induced pulmonary inflammation and mucus hyperproduction, and suggests that it may be a novel target for treatment of airway disorders or disease exacerbations with airborne particulate pollution.

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Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽

10.3390/cells8080934 ◽

2019 ◽

Vol 8 (8) ◽

pp. 934 ◽

Cited By ~ 2

Author(s):

Ecke ◽

Lutter ◽

Scholka ◽

Hansch ◽

Becker ◽

...

Keyword(s):

Articular Chondrocytes ◽

Culture Conditions ◽

Serum Supplement ◽

3 Dimensional ◽

The Matrix ◽

Differentiation Degree ◽

Overlay Technique ◽

Fetal Bovine ◽

Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

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Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

Journal of Applied Physiology ◽

10.1152/jappl.1991.70.5.2259 ◽

1991 ◽

Vol 70 (5) ◽

pp. 2259-2267 ◽

Cited By ~ 12

Author(s):

M. Somerville ◽

P. S. Richardson ◽

A. Rutman ◽

R. Wilson ◽

P. J. Cole

Keyword(s):

Pseudomonas Aeruginosa ◽

Alkaline Protease ◽

Periodic Acid ◽

Surface Epithelium ◽

Dependent Manner ◽

Periodic Acid Schiff ◽

Concentration Dependent Manner ◽

Human Bronchus ◽

Secretion Inhibition

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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Dopamine promotes colonic mucus secretion through dopamine D5 receptor in rats

AJP Cell Physiology ◽

10.1152/ajpcell.00261.2017 ◽

2019 ◽

Vol 316 (3) ◽

pp. C393-C403 ◽

Cited By ~ 4

Author(s):

Yun Li ◽

Yue Zhang ◽

Xiao-Li Zhang ◽

Xiao-Yan Feng ◽

Chen-Zhe Liu ◽

...

Keyword(s):

Transgenic Mice ◽

Colonic Mucosa ◽

Periodic Acid ◽

Mucus Secretion ◽

Mucosal Barrier ◽

Enzyme Linked Immunosorbent Assay ◽

Intracellular Camp ◽

Periodic Acid Schiff ◽

Dopamine Content

Dopamine regulates gastrointestinal mucosal barrier. Mucus plays important roles in the protection of intestinal mucosa. Here, the regulatory effect of dopamine on rat colonic mucus secretion was investigated. RT-PCR, immunofluorescence, Periodic Acid-Schiff reagent assay, Alcian blue-Periodic Acid-Schiff staining, and enzyme-linked immunosorbent assay were used to observe the expression of dopamine receptor and the direct effect of dopamine on the colonic mucus. Mice injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) destroying enteric dopamine (DA) neurons, rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra damaging central dopaminergic neurons, and dopamine D5 receptor-downregulated transgenic mice were used to detect the effect of endogenous enteric dopamine or dopamine receptors on distal colonic mucus. Our results indicated that D5 immunoreactivity was widely distributed on the colonic goblet cells. Dopamine dose-dependently increased rat distal colonic mucus secretion in vitro. D1-like receptor antagonist SCH23390 inhibited dopamine (1 μΜ)-induced distal colonic mucus secretion. D1-like receptor agonist SKF38393 promoted mucin 2 (MUC2) secretion and increased the intracellular cAMP level of colonic mucosa. D5 receptor-downregulated transgenic mice showed a decreased colonic MUC2 content. MPTP-treated mice exhibited lower colonic dopamine content and decreased colonic mucus content. 6-OHDA rats had an increase in the dopamine content in colonic mucosa but decreases in the protein levels of D1 and D5 receptors and MUC2 content in the colonic mucosa. These findings reveal that dopamine is able to promote distal colonic mucus secretion through the D5 receptor, which provides important evidence to better understand the possible role of dopamine in the colonic mucosal barrier.

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Effect of pepsin on partially purified pig gastric mucus and purified mucin

Biochemistry and Cell Biology ◽

10.1139/o88-044 ◽

1988 ◽

Vol 66 (5) ◽

pp. 367-373 ◽

Cited By ~ 4

Author(s):

Sum P. Lee ◽

Jane F. Nicholls ◽

Anthony M. Roberton ◽

Han Z. Park

Keyword(s):

Periodic Acid ◽

Gastric Mucin ◽

Low Density ◽

Gastric Mucus ◽

Experimental Conditions ◽

Periodic Acid Schiff ◽

Pepsin Digestion ◽

Cscl Density Gradient ◽

Peptic Digestion

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid – Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (<10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.

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Histochemical properties of cartilage proteoglycans.

Journal of Histochemistry & Cytochemistry ◽

10.1177/31.1.6187804 ◽

1983 ◽

Vol 31 (1) ◽

pp. 53-61 ◽

Cited By ~ 18

Author(s):

E Ippolito ◽

V A Pedrini ◽

A Pedrini-Mille

Keyword(s):

Alcian Blue ◽

Magnesium Chloride ◽

Periodic Acid ◽

Keratan Sulfate ◽

Periodic Acid Schiff ◽

Histological Sections ◽

Connective Tissue Matrix ◽

Cartilage Proteoglycans ◽

Tissue Matrix

Proteoglycan interaction with alcian blue at different concentrations of magnesium chloride was studied both in vitro and in histological sections of paraffin-embedded tissues. Our experiments indicate that a) proteoglycans with different contents of chondroitin sulfate and keratan sulfate, prepared under nondegradative conditions, are not distinguishable on the basis of the critical electrolyte concentrations at which staining is abolished; b) the state of aggregation of proteoglycans only very slightly affects the alcian blue affinity of the macromolecules at different concentrations of magnesium chloride; c) the interaction of proteoglycans with other components of the connective tissue matrix is an important factor in determining the strength of binding of alcian blue to the polyanionic macromolecules in histological sections. These factors should be considered in interpreting histochemical data obtained by staining tissue sections with alcian blue at different concentrations of magnesium chloride. Proteoglycans, like glycosaminoglycans, are only weakly periodic acid-Schiff-positive.

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Mitochondria-Targeted Antioxidant Mito-Tempo Protects Against Aldosterone-Induced Renal Injury In Vivo

Cellular Physiology and Biochemistry ◽

10.1159/000485287 ◽

2017 ◽

Vol 44 (2) ◽

pp. 741-750 ◽

Cited By ~ 14

Author(s):

Wei Ding ◽

Tingyan Liu ◽

Xiao Bi ◽

Zhiling Zhang

Keyword(s):

Electron Microscopy ◽

Renal Function ◽

Nlrp3 Inflammasome ◽

Renal Injury ◽

Periodic Acid ◽

Inflammasome Activation ◽

Periodic Acid Schiff ◽

Nlrp3 Inflammasome Activation

Background/Aims: Growing evidence suggests mitochondrial dysfunction (MtD) and the Nlrp3 inflammasome play critical roles in chronic kidney disease (CKD) progression. We previously reported that Aldosterone (Aldo)-induced renal injury in vitro is directly caused by mitochondrial reactive oxygen species (mtROS)-mediated activation of the Nlrp3 inflammasome. Here we aimed to determine whether a mitochondria-targeted antioxidant (Mito-Tempo) could prevent Aldo-induced kidney damage in vivo. Methods: C57BL/6J mice were treated with Aldo and/or Mito-Tempo (or ethanol as a control) for 4 weeks. Renal injury was evaluated by Periodic Acid-Schiff reagent or Masson’s trichrome staining and electron microscopy. ROS were measured by DCFDA fluorescence and ELISA. MtD was determined by real-time PCR and electron microscopy. Activation of the Nlrp3 inflammasome and endoplasmic reticulum stress (ERS) was detected via western blot. Results: Compared with control mice, Aldo-infused mice showed impaired renal function, increased mtROS production and MtD, Nlrp3 inflammasome activation, and elevated ERS. We showed administration of Mito-Tempo significantly improved renal function and MtD, and reduced Nlrp3 inflammasome activation and ERS in vivo. Conclusion: Mitochondria-targeted antioxidants may attenuate Aldo-infused renal injury by inhibiting MtD, the Nlrp3 inflammasome, and ERS in vivo. Therefore, targeting mtROS might be an effective strategy for preventing CKD.

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