Development of fibroblast/endothelial cell-seeded collagen scaffolds for in vitro prevascularization.

The development of vascularized scaffolds remains one of the major challenges in tissue engineering, and co-culturing with endothelial cells is known as one of the possible approaches for this purpose. In this approach, optimization of cell culture conditions, scaffolds, and fabrication techniques is needed to develop tissue equivalents that will enable in vitro formation of a capillary network. Prevascularized equivalents will be more physiologically comparable to the native tissues and potentially prevent insufficient vascularization after implantation. This study aimed to culture human umbilical vein endothelial cells (HUVECs), alone or in co-culture with fibroblasts, on collagen scaffolds prepared by simple fabrication approaches for in vitro prevascularization. Different concentrations and ratios of HUVECs and fibroblasts seeded on collagen gel and sponge scaffolds under several culture conditions were examined. Cell viability, scaffolds morphology, and structure were analyzed. Collagen gel scaffolds showed good cell proliferation and viability, with higher proliferation rates for cells cultured in a 2:1 (fibroblasts: HUVECs) ratio and kept in endothelial cell growth medium. However, these matrices were unable to support endothelial cell sprouting. Collagen sponges were highly porous and showed good cell viability. However, they became fragile over time in culture, and they still lack signs of vascularization. Collagen scaffolds were a good platform for cell growth and viability. However, under the experimental conditions of this study, the HUVEC/fibroblast-seeded scaffolds were not suitable platforms to generate in vitro prevascularized equivalents. Our findings will be a valuable starting point to optimize culture microenvironments and scaffolds during fabrication of prevascularized scaffolds.

Oral mucosa equivalents, prevascularization approaches, and potential applications.

BACKGROUND: Oral mucosa equivalents (OMEs) have been used as in vitro models (eg, for studies of human oral mucosa biology and pathology, toxicological and pharmacological tests of oral care products), and clinically to treat oral defects. However, the human oral mucosa is a highly vascularized tissue and implantation of large OMEs can fail due to a lack of vascularization. To develop equivalents that better resemble the human oral mucosa and increase the success of implantation to repair large-sized defects, efforts have been made to prevascularize these constructs. PURPOSE: The aim of this narrative review is to provide an overview of the human oral mucosa structure, common approaches for its reconstruction, and the development of OMEs, their prevascularization, and in vitro and clinical potential applications. STUDY SELECTION: Articles on non-prevascularized and prevascularized OMEs were included, since the development and applications of non-prevascularized OMEs are a foundation for the design, fabrication, and optimization of prevascularized OMEs. CONCLUSIONS: Several studies have reported the development and in vitro and clinical applications of OMEs and only a few were found on prevascularized OMEs using different approaches of fabrication and incorporation of endothelial cells, indicating a lack of standardized protocols to obtain these equivalents. However, these studies have shown the feasibility of prevascularizing OMEs and their implantation in animal models resulted in enhanced integration and healing. Vascularization in tissue equivalents is still a challenge, and optimization of cell culture conditions, biomaterials, and fabrication techniques along with clinical studies is required.

Regulation of eukaryotic translation initiation factor 6 dynamics through multisite phosphorylation by GSK3.

Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation. Both GSK3α and GSK3ß phosphorylate human eIF6. Multiple residues in the C terminus of eIF6 are phosphorylated by GSK3 in a sequential manner. In response to serum starvation, eIF6 accumulates in the cytoplasm, and this altered localization depends on phosphorylation by GSK3. Disruption of eIF6 phosphorylation exacerbates the translation inhibitory response to serum starvation and stalls cell growth. These results suggest that eIF6 regulation by GSK3 contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.

Experimental models and methods for cutaneous wound healing assessment.

Wound healing studies are intricate, mainly because of the multifaceted nature of the wound environment and the complexity of the healing process, which integrates a variety of cells and repair phases, including inflammation, proliferation, reepithelialization and remodelling. There are a variety of possible preclinical models, such as in mice, rabbits and pigs, which can be used to mimic acute or impaired for example, diabetic and nutrition-related wounds. These can be induced by many different techniques, with excision or incision being the most common. After determining a suitable model for a study, investigators need to select appropriate and reproducible methods that will allow the monitoring of the wound progression over time. The assessment can be performed by non-invasive protocols such as wound tracing, photographic documentation (including image analysis), biophysical techniques and/or by invasive protocols that will require wound biopsies. In this article, we provide an overview of some of the most often needed and used: (a) preclinical/animal models including incisional, excisional, burn and impaired wounds; (b) methods to evaluate the healing progression such as wound healing rate, wound analysis by image, biophysical assessment, histopathological, immunological and biochemical assays. The aim is to help researchers during the design and execution of their wound healing studies.

Understanding the antimicrobial activity of selected disinfectants against methicillin-resistant Staphylococcus aureus (MRSA).

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections in homes and healthcare environments. Although disruption of cytoplasmic membrane integrity has been documented as the main bactericidal effect of biocides, little is known about the biochemical alterations induced by these chemical agents. In this study, we used Fourier transform infrared (FT-IR) spectroscopy and chemometric tools as an alternative non-destructive technique to determine the bactericidal effects of commonly used disinfectants against MRSA USA-300. FTIR spectroscopy permits a detailed characterization of bacterial reactivity, allowing an understanding of the fundamental mechanism of action involved in the interaction between bacteria and disinfectants. The disinfectants studied were ethanol 70% (N = 5), isopropanol (N = 5), sodium hypochlorite (N = 5), triclosan (N = 5) and triclocarban (N = 5). Results showed less than 5% colony forming units growth of MRSA treated with triclocarban and no growth in the other groups. Nearly 70,000 mid-infrared spectra from the five treatments and the two control (untreated; N = 4) groups of MRSA (bacteria grown in TSB and incubated at 37°C (Control I) / at ambient temperature (Control II), for 24h) were pre-processed and analyzed using principal component analysis followed by linear discriminant analysis (PCA-LDA). Clustering of strains of MRSA belonging to five treatments and the discrimination between each treatment and two control groups in MRSA (untreated) were investigated. PCA-LDA discriminatory frequencies suggested that ethanol-treated spectra are the most similar to isopropanol-treated spectra biochemically. Also reported here are the biochemical alterations in the structure of proteins, lipid membranes, and phosphate groups of MRSA produced by sodium hypochlorite, triclosan, and triclocarban treatments. These findings provide mechanistic information involved in the interaction between MRSA strains and hygiene products; thereby demonstrating the potential of spectroscopic analysis as an objective, robust, and label-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA.

Blue/violet laser inactivates methicillin-resistant Staphylococcus aureus by altering its transmembrane potential.

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue light, we investigated the changes in membrane potential and the presence of free-radical-producing photo-acceptor molecules. Bacterial cultures irradiated with one or two doses of 405nm laser light (each consisting of 121J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent species as well as the voltage sensitive dye 3,3'-Diethyloxacarbocyanine iodide. The endogenous fluorescence indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes were drastic within the first 5min after irradiation with the first dose and continued slowly after the second irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that treatment does not necessarily have to be administered over a long period of time.

Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470nm) and methicillin-resistant Staphylococcus aureus.

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibiotic resistant bacterium that leads to fatal infections; however, the mechanism of bacterial death remains unclear. In this paper, to uncover the mechanism underlying the bactericidal effect of blue light, a combination of Fourier transform infrared (FTIR) spectroscopy and chemometric tools is employed to detect the photoreactivity of MRSA and its distinctive pathway toward apoptosis after treatment. The mechanism of action of UV light and vancomycin against MRSA is also investigated to support the findings. Principal component analysis followed by linear discriminant analysis (PCA- LDA) is employed to reveal clustering of five groups of MRSA samples, namely untreated (control I), untreated and incubated at ambient air (control II), irradiated with 470nm blue light, irradiated with 253.5 UV light, and vancomycin-treated MRSA. Loadings plot from PCA-LDA analysis reveals important functional groups in proteins (1683, 1656, 1596, 1542cm-1), lipids (1743, 1409cm-1), and nucleic acids region of the spectrum (1060, 1087cm-1) that are responsible for the classification of blue light irradiated spectra and control spectra. Cluster vector plots and scores plot reveals that UV light-irradiated spectra are the most biochemically similar to blue light- irradiated spectra; however, some wavenumbers experience a shift. The shifts between blue light and UV light irradiated loadings plot at νasym PO2- band (from 1228 to 1238cm-1), DNA backbone (from 970 to 966cm-1) and base pairing vibration of DNA (from 1717 to 1712cm-1) suggest distinctive changes in DNA conformation in response to irradiation. Our findings indicate that irradiation of MRSA with 470nm light induces A-DNA cleavage and that B-DNA is more resistant to damage by blue light. Blue light and UV light treatment of MRSA are complementary and distinct from the known antimicrobial effect of vancomycin. Moreover, it is known that UV-induced cleavage of DNA predominantly targets B-DNA, which is in agreement with the FTIR findings. Overall the results suggest that the combination of light and vancomycin could be a more robust approach in treating MRSA infections.

A comparison of four methods for determining viability in human dermal fibroblasts irradiated with blue light.

INTRODUCTION: Several tests are available for assessing the viability of cells; however, there is a dearth of studies comparing the results obtained with each test. We compared the capability of four viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), neutral red, trypan blue and live/dead fluorescence), to detect potential toxicity in fibroblasts irradiated with 470nm blue light. METHODS: Cells were irradiated at 3, 55, 110 and 220J/cm(2), incubated for 24h and viability assessed using each test. RESULTS: MTT assay showed significant decreases in viability when cells were irradiated with 110 and 220J/cm(2) energy fluence (dose) (89% and 57% viable cells, respectively; p<0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p<0.0001). Neutral red assay revealed significant decrease in viability when cells were irradiated with 220J/cm(2) (84% viable cells; p=0.0008, compared to control). The live/dead fluorescence assay was less sensitive, evincing 91% and 95% viable cells after irradiation with 110 and 220J/cm(2) respectively. DISCUSSION: (1) The four assays differed in their levels of sensitivity to cell viability. (2) The adverse effect of increasing doses seems to manifest as alteration of mitochondrial metabolism, followed by lysosomal dysfunction, membrane disruption and finally loss of cell membrane integrity. (3) Overall, irradiation with 3J/cm(2) or 55J/cm(2) did not adversely affect cell viability. Thus, doses below 110J/cm(2) appear safe.

Blue 470 nm light suppresses the growth of Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

BACKGROUND AND OBJECTIVE: Emerging evidence suggests that blue light can photo-inactivate some bacteria of clinical importance. Consequently, we tested the hypothesis that 470 nm light can suppress growth of two recalcitrant bacteria, MRSA and Salmonella. MATERIALS AND METHODS: We plated 5 × 106 and 7 × 106 CFU/ml USA300 strain of MRSA and 1 × 106 CFU/ml of Salmonella enterica serovars Typhimurium and Heidelberg. Plates were irradiated with 55, 110, 165 and 220 J/cm2 of blue light, incubated at 37°C for 24 hours and colony counts determined. RESULTS: Compared with controls, blue light irradiation produced a significant dose-dependent reduction in the number of colonies formed by each bacterial strain (P < 0.001). Irradiation of 5 × 106 and 7 × 106 CFU/ml MRSA with 55 J/cm2 produced 92% (4.6 × 106 CFU/ml) and 86% (6 × 106 CFU/ml) inactivation respectively, while 110 and 220 J/cm2 suppressed each MRSA density 100%. Irradiation of Salmonella Typhimurium with 55 and 110 J/cm2 suppressed bacterial growth 31% (3.1 × 105 CFU/ml) and 93% (9.3 × 105 CFU/ml) respectively; while Salmonella Heidelberg was inhibited 11% (1.1 × 105 CFU/ml) and 84% (8.4 × 105 CFU/ml) respectively by the two fluences. Complete inactivation of each Salmonella strain was achieved using 165 or 220 J/cm2 . CONCLUSION: The observed inhibition of Gram-positive (MRSA) and Gram-negative (Salmonella) bacteria suggests the versatility of blue light in bacteria eradication, making it a viable intervention strategy for decontamination of food and environments that harbor such bacteria. Lasers Surg. Med. 47:595-601, 2015. © 2015 Wiley Periodicals, Inc.

Whole-Genome Sequence for Methicillin-Resistant Staphylococcus aureus Strain ATCC BAA-1680.

We report here the whole-genome sequence of the USA300 strain of methicillin-resistant Staphylococcus aureus (MRSA), designated ATCC BAA-1680, and commonly referred to as community-associated MRSA (CA-MRSA). This clinical MRSA isolate is commercially available from the American Type Culture Collection (ATCC) and is widely utilized as a control strain for research applications and clinical diagnosis. The isolate was propagated in ATCC medium 18, tryptic soy agar, and has been utilized as a model S. aureus strain in several studies, including MRSA genetic analysis after irradiation with 470-nm blue light.

Optimization of the antimicrobial effect of blue light on methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

BACKGROUND AND OBJECTIVE: In previous studies, we showed that irradiation with 405 nm or 470 nm light suppresses up to 92% methicillin-resistant Staphylococcus aureus (MRSA) growth in vitro and that the remaining bacteria re-colonize. In this study, the aim was to develop a protocol that yields 100% MRSA growth suppression. MATERIALS AND METHODS: We cultured 3 × 10(6) and 5 × 10(6) CFU/ml USA300 strain of MRSA and then irradiated each plate with varying fluences of 1-60 J/cm2 of 405 nm or 470 nm light, either once or twice at 6 hours intervals. Next, we plated 7 × 10(6) CFU/ml and irradiated it with 45, 50, 55, or 60 J/cm2 fluence, once, twice, or thrice at the same 6 hours intervals. In a third experiment, the same culture density was irradiated with 0, 165, 180, 220, or 240 J/cm(2) , either once, twice, or thrice. RESULTS: Irradiation with either wavelength significantly reduced the bacterial colonies regardless of bacterial density (P < 0.05). At 3 × 10(6) CFU/ml density, nearly 40% and 50% growth of MRSA were suppressed with as little as 3 J/cm2 of 405 nm and 470 nm wavelengths, respectively. Moreover, 100% of the colonies were suppressed with a single exposure to 55 or 60 J/cm2 of 470 nm light or double treatment with 50, 55, or 60 J/cm2 of 405 nm wavelength. At 5 × 10(6) CFU/ml density, irradiating twice with 50, 55, or 60 J/cm2 of either wavelength suppressed bacterial growth completely, lower fluences did not. The denser 7 × 10(6) CFU/ml culture required higher doses to achieve 100% suppression, either one shot with 220 J/cm2 of 470 nm light or two shots of the same dose using 405 nm. CONCLUSION: The bactericidal effect of blue light can be optimized to yield 100% bacterial growth suppression, but with relatively high fluences for dense bacterial cultures, such as 7 × 10(6) CFU/ml.

Wavelength and bacterial density influence the bactericidal effect of blue light on methicillin-resistant Staphylococcus aureus (MRSA).

OBJECTIVE: The purpose of this study was to investigate the effect of wavelength and methicillin-resistant Staphylococcus aureus (MRSA) density on the bactericidal effect of 405 and 470 nm light. BACKGROUND DATA: It is recognized that 405 and 470 nm light-emitting diode (LED) light kill MRSA in standard 5 × 10(6) colony-forming units (CFU)/mL cultures; however, the effect of bacterial density on the bactericidal effect of each wavelength is not known. METHODS: In three experiments, we cultured and plated US300 MRSA at four densities. Then, we irradiated each plate once with either wavelength at 0, 1, 3, 45, 50, 55, 60, and 220 J/cm(2). RESULTS: Irradiation with either wavelength reduced bacterial colonies at each density (p<0.05). More bacteria were cleared as density increased; however, the proportion of colonies cleared, inversely decreased as density increased--the maximum being 100%, 96%, and 78% for 3 × 10(6), 5 × 10(6), and 7 × 10(6) CFU/mL cultures, respectively. Both wavelengths had similar effects on the sparser 3 × 10(6) and 5 × 10(6) CFU/mL cultures, but in the denser 7 × 10(6) CFU/mL culture, 405 nm light cleared more bacteria at each fluence (p<0.001). To determine the effect of beam penetration, denser 8 × 10(6) and 12 × 10(6) CFU/mL culture plates were irradiated either from the top, the bottom, or both directions. More colonies were eradicated from plates irradiated from top and bottom, than from plates irradiated from top or bottom at the same sum total fluences (p<0.001). CONCLUSIONS: The bactericidal effect of LED blue light is limited more by light penetration of bacterial layers than by bacterial density per se.