Effect of Substrate Surface Modification on Biomineralization of Osteoblasts

ABSTRACTUnderstanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the development of a successfully engineered bone tissue scaffold, and to date there has not been a well-established method for the quantitative examination of bone mineralization in situ. We investigated the mechanical properties of MC3T3-E1 osteoblast-like cells and the crystalline properties of their biomineralized ECM in vitro using shear modulation force microscopy (SMFM), confocal laser scanning microscopy (CLSM), synchrotron X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The elastic modulus of the mineralizing cells increased at time points corresponding to mineral production, whereas that of the non-mineralizing cells did not vary significantly over time. CLSM showed a restructuring of the F-actin fiber network of mineralizing cells with time, which indicates remodeling activities in the cytoskeleton and was not seen in the non-mineralizing cells. Both XRD and FTIR showed that the mineralizing subclone produced hydroxyapatite in situ and that the non-mineralizing subclone was in fact weakly biomineralizing.

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Effects of antimicrobial agents on oral biofilms in a saliva-conditioned flowcell

Biofilms ◽

10.1017/s1479050503001017 ◽

2004 ◽

Vol 1 (1) ◽

pp. 5-12 ◽

Cited By ~ 15

Author(s):

J. S. Foster ◽

P. C. Pan ◽

P. E. Kolenbrander

Keyword(s):

Laser Scanning ◽

Antimicrobial Agents ◽

Cetylpyridinium Chloride ◽

Oral Bacteria ◽

Laser Scanning Microscopy ◽

Cellular Damage ◽

Confocal Laser ◽

Oral Biofilms

Oral bacteria form mixed-species biofilms known as dental plaque. Growth of these complex microbial communities is often controlled with the use of antimicrobial mouthrinses. Novel laboratory methods for testing the efficacy of antimicrobials in situ are necessary to complement current clinical testing protocols. In this study, we examined the effects of antimicrobial agents on a streptococcal biofilm grown in a saliva-conditioned flowcell. The flowcell coupled with confocal laser scanning microscopy enabled examination of growing oral biofilms in situ without disruption of the microbial community. Biofilms composed of Streptococcus gordonii DL1 were grown in an in vitro flowcell and treated with several commercially available antimicrobial mouthrinses containing essential oils, triclosan, cetylpyridinium chloride/domiphen or chlorhexidine. The results of this study revealed varying abilities of the antimicrobial agents to cause cellular damage on the growing biofilm in situ. This study therefore demonstrated the usefulness of the flowcell in the rapid assessment of antimicrobial efficacy.

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The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures

Scientific Reports ◽

10.1038/s41598-021-86399-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Teresa Faddetta ◽

Loredana Abbate ◽

Pasquale Alibrandi ◽

Walter Arancio ◽

Davide Siino ◽

...

Keyword(s):

Laser Scanning ◽

Fungal Endophytes ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Citrus Limon ◽

Plant Microbiota ◽

Aseptic Conditions ◽

First Time

AbstractCitrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

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3-D imaging of cells using a confocal laser scanning microscope and digital image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102560 ◽

1988 ◽

Vol 46 ◽

pp. 96-97

Author(s):

Thomas M. Jovin ◽

Michel Robert-Nicoud ◽

Donna J. Arndt-Jovin ◽

Thorsten Schormann

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Confocal Laser ◽

Scanning Microscope ◽

Laser Scanning Microscope ◽

Biochemical Processes ◽

Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

Biomedical glasses ◽

10.1515/bglass-2019-0008 ◽

2019 ◽

Vol 5 (1) ◽

pp. 85-97

Author(s):

Nusrat Sharmin ◽

Mohammad S. Hasan ◽

Md. Towhidul Islam ◽

Chengheng Pang ◽

Fu Gu ◽

...

Keyword(s):

Dissolution Rate ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ion Release ◽

Scanning Calorimetry ◽

Cell Culture Study ◽

Borophosphate Glasses ◽

Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

Scientific Reports ◽

10.1038/s41598-021-87922-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Beatriz H. D. Panariello ◽

Justin K. Kindler ◽

Kenneth J. Spolnik ◽

Ygal Ehrlich ◽

George J. Eckert ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Root Canal ◽

Laser Scanning ◽

Confocal Imaging ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Microscopy Imaging ◽

Electromagnetic Stimulation ◽

Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

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Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces

AMB Express ◽

10.1186/s13568-021-01183-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Arashdeep Kaur ◽

Sanjeev Kumar Soni ◽

Shania Vij ◽

Praveen Rishi

Keyword(s):

Aspergillus Niger ◽

Laser Scanning ◽

Statistical Modelling ◽

Laser Scanning Microscopy ◽

Enzyme Treatment ◽

Confocal Laser ◽

Abiotic Surfaces ◽

Enzyme Cocktail ◽

Natural Variant

AbstractBiofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

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pH and Reduction Dual-Responsive Bi-Drugs Conjugated Dextran Assemblies for Combination Chemotherapy and In Vitro Evaluation

Polymers ◽

10.3390/polym13091515 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1515

Author(s):

Xiukun Xue ◽

Yanjuan Wu ◽

Xiao Xu ◽

Ben Xu ◽

Zhaowei Chen ◽

...

Keyword(s):

Combination Chemotherapy ◽

Laser Scanning ◽

Rational Design ◽

A549 Cells ◽

Chemotherapeutic Agents ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Tumor Resistance ◽

Oxidized Dextran

Polymeric prodrugs, synthesized by conjugating chemotherapeutic agents to functional polymers, have been extensively investigated and employed for safer and more efficacious cancer therapy. By rational design, a pH and reduction dual-sensitive dextran-di-drugs conjugate (oDex-g-Pt+DOX) was synthesized by the covalent conjugation of Pt (IV) prodrug and doxorubicin (DOX) to an oxidized dextran (oDex). Pt (IV) prodrug and DOX were linked by the versatile efficient esterification reactions and Schiff base reaction, respectively. oDex-g-Pt+DOX could self-assemble into nanoparticles with an average diameter at around 180 nm. The acidic and reductive (GSH) environment induced degradation and drug release behavior of the resulting nanoparticles (oDex-g-Pt+DOX NPs) were systematically investigated by optical experiment, DLS analysis, TEM measurement, and in vitro drugs release experiment. Effective cellular uptake of the oDex-g-Pt+DOX NPs was identified by the human cervical carcinoma HeLa cells via confocal laser scanning microscopy. Furthermore, oDex-g-Pt+DOX NPs displayed a comparable antiproliferative activity than the simple combination of free cisplatin and DOX (Cis+DOX) as the extension of time. More importantly, oDex-g-Pt+DOX NPs exhibited remarkable reversal ability of tumor resistance compared to the cisplatin in cisplatin-resistant lung carcinoma A549 cells. Take advantage of the acidic and reductive microenvironment of tumors, this smart polymer-dual-drugs conjugate could serve as a promising and effective nanomedicine for combination chemotherapy.

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PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.11.3738-3749.2001 ◽

2001 ◽

Vol 21 (11) ◽

pp. 3738-3749 ◽

Cited By ~ 238

Author(s):

Ulf Andersson ◽

Richard C. Scarpulla

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Peroxisome Proliferator ◽

Nuclear Respiratory Factor ◽

Nuclear Respiratory Factor 1 ◽

Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

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