scholarly journals Microstructural differences of fibrin and self-assembling peptide hydrogels in dental pulp stem cell behavior: the effect of chlorite-oxidized oxyamylose

2020 ◽  
Author(s):  
Mostafa EzEldeen ◽  
Burak Toprakhisar ◽  
Denise Murgia ◽  
Nick Smisdom ◽  
Olivier Deschaume ◽  
...  
Keyword(s):  
Dental Pulp ◽  
Microstructural Stability ◽  
Microscopy Imaging ◽  
Force Microscopy ◽  
Regenerative Endodontics ◽  
Self Assembling ◽  
Atomic Force ◽  
Peptide Hydrogels

Abstract Tailored hydrogels mimicking the native extracellular environment could aid in overcoming the high variability in regenerative endodontics outcomes. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. Further, to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of dental pulp stem cells (DPSCs).Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness and alignment) and by nanoindentation to measure their stiffness (elastic modulus). DPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity was determined using confocal microscopy imaging, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level, while affecting the SAP hydrogel microstructure (homogeneity) leading to fiber aggregation. The stiffness of the SAP hydrogels was 7-fold higher than the fibrin hydrogels. The viability and attachment of DPSCs and DNA content was significantly higher in fibrin hydrogels than in SAP hydrogels. The microstructural stability after COAM inclusion and the favorable DPSCs’ response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and for application in endodontic regeneration.

scholarly journals Chlorite oxidized oxyamylose differentially influences the microstructure of fibrin and self assembling peptide hydrogels as well as dental pulp stem cell behavior

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mostafa EzEldeen ◽  
Burak Toprakhisar ◽  
Denise Murgia ◽  
Nick Smisdom ◽  
Olivier Deschaume ◽  
...  
Keyword(s):  
Dental Pulp ◽  
Microstructural Stability ◽  
Force Microscopy ◽  
Regenerative Endodontics ◽  
Self Assembling ◽  
Atomic Force ◽  
Human Dental Pulp ◽  
Peptide Hydrogels

AbstractTailored hydrogels mimicking the native extracellular environment could help overcome the high variability in outcomes within regenerative endodontics. This study aimed to evaluate the effect of the chemokine-binding and antimicrobial polymer, chlorite-oxidized oxyamylose (COAM), on the microstructural properties of fibrin and self-assembling peptide (SAP) hydrogels. A further goal was to assess the influence of the microstructural differences between the hydrogels on the in vitro behavior of human dental pulp stem cells (hDPSCs). Structural and mechanical characterization of the hydrogels with and without COAM was performed by atomic force microscopy and scanning electron microscopy to characterize their microstructure (roughness and fiber length, diameter, straightness, and alignment) and by nanoindentation to measure their stiffness (elastic modulus). Then, hDPSCs were encapsulated in hydrogels with and without COAM. Cell viability and circularity were determined using confocal microscopy, and proliferation was determined using DNA quantification. Inclusion of COAM did not alter the microstructure of the fibrin hydrogels at the fiber level while affecting the SAP hydrogel microstructure (homogeneity), leading to fiber aggregation. The stiffness of the SAP hydrogels was sevenfold higher than the fibrin hydrogels. The viability and attachment of hDPSCs were significantly higher in fibrin hydrogels than in SAP hydrogels. The DNA content was significantly affected by the hydrogel type and the presence of COAM. The microstructural stability after COAM inclusion and the favorable hDPSCs' response observed in fibrin hydrogels suggest this system as a promising carrier for COAM and application in endodontic regeneration.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Morphometric characterization of fibrinogen's αC regions and their role in fibrin self-assembly and molecular organization

Nanoscale ◽  
2017 ◽  
Vol 9 (36) ◽  
pp. 13707-13716 ◽  
Author(s):  
Anna D. Protopopova ◽  
Rustem I. Litvinov ◽  
Dennis K. Galanakis ◽  
Chandrasekaran Nagaswami ◽  
Nikolay A. Barinov ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Self Assembly ◽  
Molecular Organization ◽  
Microscopy Imaging ◽  
Force Microscopy ◽  
Atomic Force ◽  
Atomic Force Microscopy Imaging ◽  
Morphometric Characterization

High-resolution atomic force microscopy imaging reveals the role of fibrinogen αC regions in the early stages of fibrin self-assembly.

scholarly journals A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

2017 ◽  
Vol 96 (9) ◽  
pp. 1051-1057 ◽  
Author(s):  
A.C. Santiago ◽  
Z.N. Khan ◽  
M.C. Miguel ◽  
C.C. Gironda ◽  
A. Soares-Costa ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Surface Hardness ◽  
Dental Enamel ◽  
Reversible Inhibitors ◽  
Force Microscopy ◽  
Atomic Force ◽  
Enamel Erosion ◽  
Acquired Enamel Pellicle

Cystatin B was recently identified as an acid-resistant protein in acquired enamel pellicle; it could therefore be included in oral products to protect against caries and erosion. However, human recombinant cystatin is very expensive, and alternatives to its use are necessary. Phytocystatins are reversible inhibitors of cysteine peptidases that are found naturally in plants. In plants, they have several biological and physiological functions, such as the regulation of endogenous processes, defense against pathogens, and response to abiotic stress. Previous studies performed by our research group have reported high inhibitory activity and potential agricultural and medical applications of several sugarcane cystatins, including CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4. In the present study, we report the characterization of a novel sugarcane cystatin, named CaneCPI-5. This cystatin was efficiently expressed in Escherichia coli, and inhibitory assays demonstrated that it was a potent inhibitor of human cathepsins B, K, and L ( Ki = 6.87, 0.49, and 0.34 nM, respectively). The ability of CaneCPI-5 to bind to dental enamel was evaluated using atomic force microscopy. Its capacity to protect against initial enamel erosion was also tested in vitro via changes in surface hardness. CaneCPI-5 showed a very large force of interaction with enamel (e.g., compared with mucin and casein) and significantly reduced initial enamel erosion. These results suggest that the inclusion of CaneCPIs in dental products might confer protection against enamel erosion.

scholarly journals Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

2016 ◽  
Vol 397 (5) ◽  
pp. 401-415 ◽  
Author(s):  
Sara Cappelli ◽  
Amanda Penco ◽  
Benedetta Mannini ◽  
Roberta Cascella ◽  
Mark R. Wilson ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Aggregate Size ◽  
Solvent Exposure ◽  
Microscopy Imaging ◽  
N Protein ◽  
Force Microscopy ◽  
Fluorescence Measurements ◽  
Atomic Force ◽  
Αb Crystallin

Abstract Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

Controlling neuronal growth and connectivity via directed self-assembly of proteins

2013 ◽  
Vol 1498 ◽  
pp. 207-212
Author(s):  
Daniel Rizzo ◽  
Ross Beighley ◽  
James D. White ◽  
Cristian Staii
Keyword(s):  
Self Assembly ◽  
Gold Surface ◽  
Nerve Tissue ◽  
3 Dimensional ◽  
Force Microscopy ◽  
Self Assembling ◽  
Atomic Force ◽  
Will Self ◽  
Neuron Growth

ABSTRACTMaterials that offer the ability to influence tissue regeneration are of vital importance to the field of Tissue Engineering. Because valid 3-dimensional scaffolds for nerve tissue are still in development, advances with 2-dimensional surfaces in vitro are necessary to provide a complete understanding of controlling regeneration. Here we present a method for controlling nerve cell growth on Au electrodes using Atomic Force Microscopy -aided protein assembly. After coating a gold surface in a self-assembling monolayer of alkanethiols, the Atomic Force Microscope tip can be used to remove regions of the self-assembling monolayer in order to produce well-defined patterns. If this process is then followed by submersion of the sample into a solution containing neuro-compatible proteins, they will self assemble on these exposed regions of gold, creating well-specified regions for promoted neuron growth.

Distribution of sulphated polysaccharides within calcareous biominerals suggests a widely shared two-step crystallization process for the microstructural growth units

2008 ◽  
Vol 72 (1) ◽  
pp. 233-237 ◽  
Author(s):  
J. P. Cuif ◽  
Y. Dauphin ◽  
B. Farre ◽  
G. Nehrke ◽  
J. Nouet ◽  
...  
Keyword(s):  
Crystallization Process ◽  
Growth Layer ◽  
Microscopy Imaging ◽  
Sulphated Polysaccharides ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
Vital Effects ◽  
Ca Carbonate

AbstractSynchrotron-based XANES characterization of sulphated sulphur combined with atomic force microscopy and transmission electron microscopy (imaging and diffraction) allow insights into the crystallization of the calcareous units produced by invertebrates. As a result of a series of converging data, reticulate crystallization of the amorphous Ca-carbonate molecules conveyed to the micron-thick growth layer by the sumicrometric organo-mineral units seems a reasonable hypothesis, providing us with a method of explaining the multiple and taxonomy-linked ‘vital effects’ which have long been recognized among the calcareous biocrystals.

scholarly journals Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro

2021 ◽  
Vol 11 ◽  
Author(s):  
Kaila Petronila Medina-Alarcón ◽  
Iara Pengo Tobias da Silva ◽  
Giovana Garcia Ferin ◽  
Marcelo A. Pereira-da-Silva ◽  
Caroline Maria Marcos ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Paracoccidioides Brasiliensis ◽  
Public Health Problem ◽  
Extracellular Polysaccharide ◽  
Force Microscopy ◽  
Atomic Force ◽  
High Degree ◽  
First Time

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3’hydroxychalcone (3’chalc) -loaded nanoemulsion (NE) (NE3’chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3’chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3’hidroxychalcone (3’chalc) range from 0.97- 7.8 µg/mL and NE3’chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3’chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3’chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3’chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

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