Number Of Adherent Cells Research Articles

Physicochemical evaluation of poultry farming materials for bacterial adhesion and biofilm formation: implications for contamination prevention

ABSTRACT In poultry production, various substrates and the adhesive properties of pathogenic bacteria lead to biofilm formation and bio-contamination. This study investigates bacterial attachment behaviour on materials commonly used in poultry farming to aid in selecting appropriate materials and mitigating bio-contamination risks. We compared the hygienic profiles of these materials and elucidated the physicochemical mechanisms governing adhesion. Physicochemical properties and surface interaction energies between Escherichia coli ATCC25922 and five substrates were estimated using the contact angle method, and the number of adherent cells was quantified experimentally. Results showed that all substrates, except for the underside of straw (SUS), were predominantly hydrophobic. The E. coli ATCC25922 strain showed strong affinity towards polypropylene (PP) and high-density polyethylene (PEHD). The difference in adherence abilities of E. coli ATCC25922 was significantly affected by quantitative hydrophobicity and electron donor character. This study highlights factors influencing bacterial adhesion, aiding in the prevention of biofilm.

MFGE8 Acts as a Cell Adhesion Factor for Human-Induced Pluripotent Stem Cells in Embryology.

Mouse embryonic fibroblasts (MEFs) have been widely used as feeder cells in embryonic stem cell cultures because they can mimic the embryonic microenvironment. Milk fat globule-epidermal growth factor 8 (MFGE8) is expressed during mouse gonadal development, 10.5-13.5 embryonic, and is also found in MEF-conditioned medium (MEF-CM). Feeder-less culture of human-induced pluripotent stem cells (iPSCs) with MEF-CM significantly decreased the number of adherent cells when an inhibitory antibody against MFGE8 was used. The concentration of mouse MFGE8 in MEF-CM, as measured by an ELISA (Enzyme-Linked Immunosorbent Assay), was 0.16-1.24 μg/mL. Mouse MFGE8 and human MFGE8 have partially different molecular structures. Both the recombinant mouse MFGE8 and human MFGE8 significantly promoted cell adhesion of human iPSCs at medium-added concentrations of 2 μg/mL. This cell adhesion was also strongly inhibited by Arginylglycylaspartic acid (RGD) inhibitors, suggesting that it is dependent on the RGD sequence. The integrin αVβ5 expressed in iPSCs was thought to be involved in binding to the RGD sequence. MEF-CMs have long been an essential bio-derived material for the feeder culture method of iPSC culture. This study demonstrates that MFGE8 in MEF-CM is a functional factor in the promoting of cell adhesion of human iPSCs. Furthermore, the use of MFGE8-containing media demonstrates that iPSCs can be established and cultured while maintaining pluripotency and inducing three germ layer differentiation. The results of this study suggest the possibility of using MFGE8 as a scaffold material suitable for inducing differentiation when reproducing invivo maturation in vitro.

CCQM-P123 number and geometric property of cells adhered to a solid substrate

Main textSeveral scientific fields, such as cytology, biochemistry, molecular biology and genetics have cells as their direct topic of study. The cell is a fundamental unit of highly technological systems, employed, for example, in the development of new chemicals and drugs, in the evaluation of compound toxicity, in novel approaches to health, such as regenerative medicine. High content screening technologies are cellular imaging-based assays. In this framework, cell density, cell confluency and cell size are measurands that can describe a biological effect of a molecule under examination. The CCQM-P123 study aimed to demonstrate capability of participants in quantifying 4 measurands using one reference material: number of adherent cells on a planar surface, cell density in the range 0 to 5000 cells/mm2, cell confluency in the range 0 % to 100 % and average cell area. Nine Institutions participated in the study. Reference materials for three levels of cell density and cell confluency were prepared for fluorescence microscopy and circulated among participants. Each reference material was a commercial imaging dish having 400 squares and seeded with human cells, fixed and stained for nucleus and for whole cell. On each dish 4 squares were selected for the analysis as "measurement standards". The use of a calibrated ruler to define the area of measurement assured the traceability to the International System of Units (SI). Results for the 4 measurands showed an overall good agreement between the laboratories. The study allowed fundamental considerations in the frame of a cell-based measurement comparison, in terms of: traceability, comparability of results and complexity of the study protocol. The study has been a preparatory exercise for a key comparison on at least one of the 3 measurements claims (cell density, cell confluency and cell area) related to adhered cells features such as cell proliferation, morphology and size.To reach the main text of this paper, click on Final Report.The final report has been peer-reviewed and approved for publication by the CCQM.

Antibiofilm mechanism of mouse gastrointestinal stimulation against Vibrio parahaemolyticus under bile salt culture.

Bile salts are crucial microbe-selective inhibitors present in the intestinal tracts of humans and other animals. Environmental and clinical strains of Vibrio parahaemolyticus (V. parahaemolyticus) exhibited different biofilm-forming abilities under bile salt incubation. In order to find an effective way to eliminate biofilm, in this study, environmental strains were subjected to mouse gastrointestinal (GI) stimulation and cultured in medium containing 0.06% bile salts. The effects of GI stimulation on V. parahaemolyticus biofilm formation were evaluated by biofilm cells assay, atomic force microscopy (AFM) assay, confocal laser scanning microscopy (CLSM) assay, extracellular polysaccharide (EPS) assay, and salmon surface biofilm formation assay. The results showed that GI stimulation diminished the ability of V. parahaemolyticus to form biofilm, significantly reduced biofilm cells, decreased the level of EPS, and destroyed the biofilm structure. For the biofilm formed by V. parahaemolyticus after GI stimulation, AFM observed that the appearance of the biofilm became inhomogeneous and rough, and CLSM observed that the 3D structure of the biofilm became dispersed and sparse. GI stimulation reduced the ability of V. parahaemolyticus to form biofilms on the surface of salmon containing 0.06% bile salts at both 12h and 24h, as evidenced by a decrease in the number of adherent cells. Comparing biofilms formed by tdh-positive V. parahaemolyticus before and after undergoing GI stimulation, a total of 1169 differentially expressed genes (DEGs) were identified by RNA sequencing. And 10 of the biofilm-related genes displayed significant down-regulation after GI stimulation. Enrichment analysis of DEGs revealed that affecting the switch between succinate and fumarate in the TCA cycle could inhibit biofilm formation. This study offers new insights into strategies for preventing biofilm formation by foodborne pathogens.

Additively manufactured porous Ti6Al4V Alloy with excellent mechanical properties, corrosion resistance, in vitro and in vivo biocompatibility

In view of the insufficient bearing capacity of porous Ti6Al4V (TC4) alloy, four kinds of porous TC4 alloys with a wall thickness of 200 μm and a side length ranging from 400 to 700 μm were designed and subsequently prepared by Additive Manufacturing (Selective Laser Melting, SLM). The results indicated that the porous TC4 alloy was primarily comprised of α' phase and a small amount of β phase. As the side length increased, the size and volume of the acicular α' phase increased while the β phase decreased. Besides, the dislocation density also increased with the increase of side length. The porous TC4 alloy with a side length of 700 μm exhibited the maximum dislocation density of 6.07×1014 m2. The yield strength and elastic modulus initially decreased and then increased with the increase of side length. Moreover, the increase in side length induced the decrease of Icorr from 1.807 × 10−6 to 4.777 × 10−7 A/cm2, especially that sample 7–2 presented the best corrosion resistance with the smallest Icorr value (4.777 × 10−7 A/cm2). It was observed by in vitro assessment that the optical density (OD) of the porous TC4 alloys surpassed 75 % on days 1, 4, and 7. It was noteworthy that the OD value of the porous TC4 alloys with a side length of 700 μm surpassed 100 % on day 7. Furthermore, the number of adherent cells displayed an increasing trend with the increase in side length. After implanting into the alveolar bone of rabbits at 12th week, the porous TC4 alloy with a side length of 700 μm induced bone growth due to a higher percentage of bone volume compared to the dental implant. After 12 weeks of in vivo experiments, the results showed that the porous TC4 alloy could reduce the inflammatory response, promote the growth of bone tissue, and have excellent osteogenic properties.

Effects of Physically Adsorbed and Chemically Immobilized RGD on Cell Adhesion to a Hydroxyapatite Surface

The strategies used to associate peptide arginylglycylaspartic acid (RGD) with calcium phosphate grafts to enhance cell–biomaterial interactions have been controversial in the literature. Several works have demonstrated that RGD-functionalized hydroxyapatite (HA) surfaces improve cell adhesion, whereas others claim that RGD-loaded HA has an inhibitory effect when serum is present in the biological medium. To investigate such contradictory results, we associated RGD with the HA surface using physical adsorption and chemical bonding methods and evaluated the cell adhesion and spreading in pre-osteoblasts culture with and without fetal bovine serum (FBS). The effect of functionalization methods on the physicochemical characteristics of both surfaces was analyzed using multiscale techniques. Adsorption assays of serum allowed us to estimate the impact of the association method on the HA surface’s reactivity. Physically adsorbed RGD did not increase the number of adhered cells due to the weak interactions between the peptide and the surface. Although chemical binding stabilizes RGD on the HA, the functionalization procedure covered the surface with molecules such as (3-aminopropyl)triethoxysilane (APTEs) and carbodiimide, changing the surface’s chemical activity. Serum protein adsorption decreased by 90%, revealing a significant reduction in the surface interactions with molecules of the biological medium. The present study’s findings showed that the RGD’s physical association with HA did not improve cell adhesion and that this phenomenon is highly dependent on the presence of serum proteins.

Efficacy of a lytic bacteriophage vB_EcoM_SQ17 against Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli biofilms on cucumber

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) and Enterotoxigenic E. coli (ETEC) have been found to readily develop biofilms on cucumber (Cucumis sativus L.), presenting a significant risk to the safety of ready-to-eat vegetables. This study aimed to assess the effectiveness of the lytic bacteriophage vB_EcoM_SQ17 (SQ17) against EHEC O157:H7 and ETEC biofilms on cucumber. Here, we evaluated the efficacy of phage SQ17 on the formation and reduction of biofilms formed by EHEC O157:H7 and ETEC strains on various surfaces, including polystyrene, poly-d-lysine precoated films, and fresh-cut cucumber, at different temperatures. Phage SQ17 significantly inhibited ETEC biofilm formation, reducing the number of adhered cells by 0.15 log CFU/mL at 37 °C. Treatment with phage SQ17 also significantly decreased the number of adhered cells in established biofilms via SEM observation. Moreover, phage SQ17 effectively reduced the biomass of EHEC O157:H7 and ETEC biofilms by over 54.8 % at 37 °C after 24 h of incubation. Following phage treatment, the viability of adhered EHEC O157:H7 cells decreased by 1.37 log CFU/piece and 0.46 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. Similarly, the viability of ETEC cells decreased by 1.07 log CFU/piece and 0.61 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. These findings suggest that phage SQ17 shows promise as a potential strategy for eradicating pathogenic E. coli biofilms on cucumber.

Optimizing small-diameter vascular grafts: Harnessing the power of recombinant human type III collagen (rhCOLIII) for enhanced antiplatelet and endothelialization performance

Small-diameter vascular grafts hold great promise as potential substitutes for coronary and peripheral arteries. However, their clinical availability is hindered by thrombogenicity and intimal hyperplasia. Herein, bilayer vascular grafts with enhanced antithrombogenicity and endothelialization were developed by two-step electrospinning technology. First, the well-designed recombinant human type III collagen (rhCOLIII), featuring numerous triplets that promote cell adhesion while excluding sequences prone to platelet adhesion and activation, was composited with poly(L-lactide-co-ε-caprolactone) (PLCL) to create a bioactive inner layer. Afterward, a pure PLCL outer layer with exceptional elasticity was constructed to provide mechanical support. In vitro antiplatelet and cytocompatibility experiments revealed that the graft containing 60% rhCOLIII exhibited almost no platelet adhesion, while the number of adhered endothelial cells increased approximately 20 times compared to pure PLCL. Furthermore, Beagle dog femoral artery implantation models demonstrated that the rhCOLIII-modified grafts underwent endothelialization within three months, contributing to the patency of grafts. The potential mechanism for enhanced endothelialization by rhCOLIII was investigated through transcriptome sequencing and verified by an integrated analysis of gene and protein expression. The cell-affinity units, Gly-Glu-Arg (GER) and Gly-Glu-Lys (GEK) triplets, present in rhCOLIII contribute to initial adhesion of endothelial cells. Following adhesion, they may upregulate the expression of integrins αVβ8 on the cell surface, thereby further enhancing cell adhesion. Subsequently, the FAK/PI3K/AKT pathway within endothelial cells may also be activated to promote cell proliferation.

Can pulsed laser treatment reduce microbial adhesion on the surface of resin denture base materials?

Objectives. The present work investigates the influence of pulsed nanosecond laser patterning of two commercial heat cure poly (methyl methacrylate) denture base materials, Trevalon and DPI heat cure, on their surface characteristics such as roughness, hydrophobicity, and microbial adhesion. Methods. A Q-switched Nd:YAG solid state nanosecond pulsed laser at a wavelength of 532 nm with a fluence of 2.55 × 1010 W cm−2 having a pulse frequency of 10 Hz was used at varying translation stage speed and vertical spacing for the surface patterning of denture base materials. The surface properties of control and patterned materials were characterized by measuring the compositional changes, wettability, and roughness. Microbial adhesion was assessed by incubating the specimens in Candida albicans suspension at 37 °C for 2 h followed by estimating the number of adherent Candida cells. Results. The micro-Raman spectroscopic studies indicated no chemical changes in the materials due to laser patterning. However, laser patterning increased the average surface roughness from 0.02 ± 0.005 and 0.04 ± 0.003 μm to 3.85 ± 0.20 μm and 3.70 ± 0.12 μm respectively for Trevalon and DPI heat cure materials. In addition, a reduction in the surface wettability was evident as manifested by an increase in water contact angle from 76 ± 2° to 105 ± 1° for Trevalon and from 72 ± 1° to 97 ± 1° for DPI heat cure. Finally, the microbial adhesion studies clearly indicated that the surfaces with higher hydrophobicity and roughness following laser patterning exhibited reduced microbial adhesion. Significance. Laser patterning can be utilized to tune the surface features of denture base materials and thus offer a promising method to create microbial adhesion resistant surfaces. Clinically, such surfaces help in reducing the incidence of denture stomatitis, especially in immunocompromised patients, without altering the composition and bulk properties of denture base materials.

The Functional Study of Response Regulator ArlR Mutants in Staphylococcus Aureus

Staphylococcus aureus is a major cause of hospital-associated infections worldwide. The organism’s ability to form biofilms has led to resistance against current treatment options such as beta-lactams, glycopeptides, and daptomycin. The ArlRS two-component system is a crucial regulatory system necessary for S. aureus autolysis, biofilm formation, capsule synthesis, and virulence. This study aims to investigate the role of the arlR deletion mutant in the detection and activation of S. aureus. We created an arlR deleted mutant and complementary strains and characterized their impact on the strains using partial growth measurement. The quantitative real-time PCR was performed to determine the expression of icaA, and the microscopic images of adherent cells were captured at the optical density of 600 to determine the primary bacterial adhesion. The biofilm formation assay was utilized to investigate the number of adherent cells using crystal violet staining. Eventually, the Triton X-100 autolysis assay was used to determine the influence of arlR on the cell autolytic activities. Our findings indicate that the deletion of arlR reduced the transcriptional expression of icaA but not icaR in the ica operon, leading to decrease in polysaccharide intercellular adhesin (PIA) synthesis. Compared to the wild-type and the complementary mutants, the arlR mutant exhibited decreased in biofilm production but increased autolysis. It concluded that the S. aureus response regulatory ArlR influences biofilm formation, agglutination, and autolysis. This work has significantly expanded our knowledge of the ArlRS two-component regulatory system and could aid in the development of novel antimicrobial strategies against S. aureus.

A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 μg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 μl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-β estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Fabrication and evaluation of composite hydroxyapatite coating on ordered micro-/nanotextured titanium surface

Objective: To develope a titanium specimen with good osteogenic activity through fabrication of a composite hydroxyapatite coating on ordered micro-/nanotextured titanium surface. Methods: An ordered micro-/nanotextured structure was prepared on the surface of titanium (the control), and then hydroxyapatite was deposited on the as-prepared ordered micro-/nanotextured structure by alternative loop immersion method. The ordered micro-/nanotextured structures before and after hydroxyapatite deposition were denoted as HA and MN, respectively. Surface morphology was observed using a scanning electron microscope. Bone marrow mesenchymal stem cells (BMMSC) were seeded on the surface of three different materials. Cell morphology was observed with a scanning electron microscope. Cell adhesion and cell proliferation were evaluated using 4', 6-diamidino-2-phenylindole staining and cell counting kit-8 assay, respectively. Extracellular matrix mineralization and the expression levels of osteogenesis-related genes were evaluated by alizarin red staining and real-time quantitative PCR, respectively. Each group has three samples in every experiment. Results: After alternative loop immersing, the MN's original microholes (20 μm in diameter) were retained, and the uniform petal-like hydroxyapatite was deposited on the MN's original titania nanotubes (70 nm in diameter). Compared with the control, BMMSC on MN and HA elongated further and intersected along the micron structure with noticeable pseudopodia and pseudoplates, and the trend was more pronounced especially on HA. The number of early adherent cells on HA was remarkably larger than that on the control and MN at each time point (P<0.05). On day 1, the A value of cell proliferation on HA was significantly higher than that on the control and MN (P<0.05). The A value of cell proliferation on HA was significantly lower than that on the control and MN on day 3 (P<0.05). On day 7, the A value of cell proliferation on HA was significantly lower than that on MN (P<0.05), but there was no statistically significant difference in the A value of cell proliferation between HA and the control on day 7 (P>0.05). The Avalue of extracellular matrix mineralization on HA (0.607±0.011) was significantly higher than that on the control and MN (0.268±0.025 and 0.522±0.022, respectively) (t=-0.25, P<0.001; t=-0.34, P<0.001). The expression levels of bone related genes on HA were significantly higher than those on the control and MN (P<0.05). Conclusions: HA could promote the BMMSC adhesion and osteogenic differentiation, support BMMSC proliferation, and demonstrate good osteogenic activity.

Differentiation Potential Analysis of Bone Marrow Mesenchymal Stem Cells from Guizhou Miniature Pigs

In this study, different growth characteristics of hematopoietic stem cells and bone marrow mesenchymal stem cells were used to isolate and culture BMSCs by whole bone marrow adhesion method, and RT-PCR was used to detect cell surface markers CD29, CD31, CD34, CD44, CD71, CD73, CD90. The differentiation of osteocytes into adipocytes and osteoblasts was induced in vitro, and the differentiation ability was observed by staining with oil red O and rubinin. To investigate the isolation and culture methods of BMSCs from Guizhou miniature pigs (Sussa lvanius) in vitro, and to study their biological characteristics and potential for multi-differentiation. The results showed that a small number of adherent cells could be seen in the primary BMSCs cultured in vitro within 48 hours, and the confluent cells reached 90% in 5~6 days. The cell growth state was good, and the shape was spindle and polygonal. RT-PCR was used to detect the positive expression of CD29, CD44, CD71, CD73 and CD90 on the cell surface, and the negative expression of CD31 and CD34. Oil red O staining showed the formation of lipid droplets in the cells, and alizarine red staining showed calcium nodules in the cells, indicating that the cells isolated and cultured in this experiment had the ability of multidirection differentiation. It is consistent with the characteristics of BMSCs. The growth of BMSCs cultured in vitro was stable, and the expanded BMSCs had multidirectional differentiation potential, which was an ideal seed cell for tissue engineering.

Perspectives on antimicrobial properties of Paulownia tomentosa Steud. fruit products in the control of Staphylococcus aureus infections

Ethnopharmacological relevancePaulownia tomentosa Steud. (P. tomentosa) is a medium-sized tree traditionally used in Chinese folk medicine for the treatment of infectious diseases. It is a rich source of prenylated phenolic compounds that have been extensively studied for their promising biological activities. Aim of the studyDue to the increasing development of antibiotic resistance, our study investigated plant-derived natural products from the fruits of P. tomentosa that could control Staphylococcus aureus infections with novel targets/modes of action and reduce antimicrobial resistance. Materials and methodsThe ethanolic extract was fractionated and detected by liquid chromatography. The antistaphylococcal effects of the plant formulations were studied in detail in vitro by various biological methods, including microdilution methods for minimum inhibitory concentration (MIC), the checkerboard titration technique for synergy assay, fluorescence measurements for membrane disruption experiments, autoinducer-2-mediated bioassay for quorum sensing inhibition, and counting of colony-forming units for relative adhesion. Morphology was examined by transmission electron microscopy. ResultsTotal ethanolic extract and chloroform fraction showed MICs of 128 and 32 μg/mL, respectively. Diplacol, diplacone, and 3′-O-methyl-5′-hydroxydiplacone inhibited S. aureus growth in the range of 8–16 μg/mL. Synergistic potential was shown in combination with mupirocin and fusidic acid. The ethanolic extract and the chloroform fraction destroyed the cell membranes by 91.61% and 79.46%, respectively, while the pure compounds were less active. The ethanolic extract and the pure compounds reduced the number of adhered cells to 47.33–10.26% compared to the untreated control. All tested plant formulations, except diplacone, inhibited quorum sensing of S. aureus. Transmission electron microscopy showed deformation of S. aureus cells. ConclusionsThe products from the fruit of P. tomentosa showed antimicrobial properties against S. aureus alone and in combination with antibiotics. By affecting intracellular targets, geranylated flavonoids proposed novel approaches in the control of staphylococcal infections.

Dual peptide functionalization of Zn alloys to enhance endothelialization for cardiovascular applications

A new generation of fully bioresorbable metallic Zn-based alloys could be used for stenting applications; however, the initial surface degradation delays stent re-endothelialization. Thus, this work proposes a dual strategy to control the corrosion and accelerate the endothelialization of ZnMg and ZnAg biodegradable alloys. First, a stable polycaprolactone (PCL) coating is obtained and followed by its functionalization with either linear RGD (Arg-Gly-Asp) or REDV (Arg-Glu-Asp-Val) peptides or a dual peptide-based platform combining both sequences (RGD-REDV). Scratching tests showed neither delamination nor detachment of the polymeric coating. Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion resistance after PCL coating by revealing lower current density and higher absolute impedance values. X-ray photoelectron spectroscopy (XPS) and fluorescent microscopy confirmed the correct peptide immobilization onto PCL coated Zn alloys. The functionalized samples exhibited enhanced human umbilical vein endothelial cells (HUVEC) adhesion. The higher number of adhered cells to the functionalized surfaces with the RGD-REDV platform demonstrates the synergistic effect of combining both RGD and REDV sequences. Higher corrosion resistance together with enhanced endothelialization indicates that the dual functionalization of Zn alloys with PCL and peptide-based RGD-REDV platform holds great potential to overcome the clinical limitations of current biodegradable metal stents.

Influence of structural parameters of 3D-printed triply periodic minimal surface gyroid porous scaffolds on compression performance, cell response, and bone regeneration.

In this study, multi-scale triply periodic minimal surface (TPMS) porous scaffolds with uniform and radial gradient distribution on pore size were printed based on the selective laser melting technology, and the influences of porosity, pore size and radial pore size distribution on compression mechanical properties, cell behavior, and bone regeneration behavior were analyzed. The results showed that the compression performance of the uniform porous scaffolds with high porosity was similar to that of cancellous bone of pig tibia, and the gradient porous scaffolds have higher elastic modulus and compressive toughness. After 4 days of cell culture, cells were distributed on the surface of scaffolds mostly, and the number of adherent cells was higher on the small pore size porous scaffolds; After 7 days, the area and density of cell proliferation on the scaffolds were improved; After 14 days, the cells on the small pore size scaffolds tended to migrate to adjacent pores. Animal implantation experiments showed that collagen fiber osteoid was intermittent on scaffolds with high porosity and large pore size, which was not conducive to bone formation. The appropriate pore size and porosity of bone regeneration were 792 um and 83%, respectively, and the regenerative ability of gradient pore size was better than that of uniform pore size. Our study explains the rules of TPMS gyroid structure parameters on compression performance, cell response and bone regeneration, and provides a reference value for the design of bone repair scaffolds for clinical orthopedics.

Quantifying Adhesion of Inflammatory Cells to the Endothelium In Vitro.

We present a simple and quantitative assay system that accurately models human endothelium by use of primary human umbilical vein endothelial cells (HUVECs) in cell culture plates coated with gelatin, a matrix that mimics basal lamina, the matrix that is tightly associated with the vascular endothelium and is critical for its proper function. We describe using this system to quantitatively measure adhesion of the inflammatory cells - monocytic THP-1 cell line to the HUVEC monolayer. The THP-1 cells are fluorescently labeled which allows to quantify the number of the fluorescent THP-1 cells adhering to the endothelium under a microscope and the level of florescence - a quantitative measure of the number of adhering fluorescent THP-1 cells using a fluorescent plate reader. After optimization, we were able to detect increased adhesion of the THP-1 cells to the endothelium in response to the inflammatory cytokine TNFα in a dose-dependent manner like what has been observed in vivo.

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells.

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Influence of High-Frequency, Low-Voltage Alternating Electric Fields on Biofilm Development Processes of Escherichia coli and Pseudomonas aeruginosa

A non-chemical solution is needed to control biofilms in water and wastewater treatment systems. High-frequency alternating electric field application offers an alternative approach that does not involve undesired electrode surface reactions. However, the effect of high-frequency alternating electric fields on bacterial cells in the attached-growth mode remains unexplored. This study investigated the impact of such fields on two stages of the biofilm development process: the initial adhesion phase (stage 1) and the early development phase (stage 2). Experiments were conducted using Escherichia coli and Pseudomonas aeruginosa in a three-channel flow cell exposed to alternating electric fields (3.05 V/cm, 20 MHz). The primary outcome of this study demonstrated that alternating electric fields decreased adhered cell numbers at both stages due to their inhibitory effect on growth. The alternating electric fields also triggered cell detachment after the initial attachment stage but not in mature biofilms. Interestingly, despite a reduction in cell counts, the amount of total biofilm biomass remained unaffected, which was likely due to increased cell size via cell elongation compensating for the decrease in numbers. No synergistic effects with respect to hydrodynamic forces were observed. These findings highlight the potential applicability of alternating electric fields to biofilm control and provide implications for water and wastewater engineering applications.