Atul A. Chaudhari

Future Prospects for Scaffolding Methods and Biomaterials in Skin Tissue Engineering: A Review

Over centuries, the field of regenerative skin tissue engineering has had several advancements to facilitate faster wound healing and thereby restoration of skin. Skin tissue regeneration is mainly based on the use of suitable scaffold matrices. There are several scaffold types, such as porous, fibrous, microsphere, hydrogel, composite and acellular, etc., with discrete advantages and disadvantages. These scaffolds are either made up of highly biocompatible natural biomaterials, such as collagen, chitosan, etc., or synthetic materials, such as polycaprolactone (PCL), and poly-ethylene-glycol (PEG), etc. Composite scaffolds, which are a combination of natural or synthetic biomaterials, are highly biocompatible with improved tensile strength for effective skin tissue regeneration. Appropriate knowledge of the properties, advantages and disadvantages of various biomaterials and scaffolds will accelerate the production of suitable scaffolds for skin tissue regeneration applications. At the same time, emphasis on some of the leading challenges in the field of skin tissue engineering, such as cell interaction with scaffolds, faster cellular proliferation/differentiation, and vascularization of engineered tissues, is inevitable. In this review, we discuss various types of scaffolding approaches and biomaterials used in the field of skin tissue engineering and more importantly their future prospects in skin tissue regeneration efforts.

Advances in Skin Regeneration Using Tissue Engineering

Tissue engineered skin substitutes for wound healing have evolved tremendously over the last couple of years. New advances have been made toward developing skin substitutes made up of artificial and natural materials. Engineered skin substitutes are developed from acellular materials or can be synthesized from autologous, allograft, xenogenic, or synthetic sources. Each of these engineered skin substitutes has their advantages and disadvantages. However, to this date, a complete functional skin substitute is not available, and research is continuing to develop a competent full thickness skin substitute product that can vascularize rapidly. There is also a need to redesign the currently available substitutes to make them user friendly, commercially affordable, and viable with longer shelf life. The present review focuses on providing an overview of advances in the field of tissue engineered skin substitute development, the availability of various types, and their application.

Multifunctionally Modified Superhydrophobic Aluminum and Fabric Surfaces with Reduced Gram-Negative and Gram-Positive Bacterial Attachment: A Possible Approach for Self-Cleaning Aircraft and Crew Cabin Surfaces

Development of novel approaches for reducing the cost of routine cleaning of aircrafts and crew cabin surfaces is a challenge. In particular, the removal of bacterial pathogens engaged in biofilm formation and multi-drug resistance is of the utmost importance. “Self-cleaning” materials such as functionally modified superhydrophobic surfaces are of a greater use as they not only remove the bacteria by their antimicrobial component, but also do not allow the bacterial attachment to the superhydrophobic surfaces, thereby limiting the biofilm formation. In the present study, we tested three surface-modified fabrics and three aluminum sheet surfaces for their bacterial attachment properties using Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) bacteria. The surface-modified fabrics were coated Nomex fabric (1382 and 1386) and uncoated Nomex fabric (1384). The aluminum sheets were coated with fluorofunctionalized copper-doped magnesium oxide (Cu–MgO) (1381 and 1434) and fluorofunctionalized silver-doped magnesium oxide (Ag–MgO) (1437). A drop of 100 μl, containing 1 × 105 colony forming units (cfu/ml) of gram-negative/gram-positive bacteria, was placed on each material and the sample with bacteria was subsequently incubated for 1 h at 37°C. After incubation, the drop was removed and the surfaces were either washed three times with phosphate buffered saline (PBS) or left unwashed. Both the washed and unwashed surfaces were immersed in 1 ml of PBS and the solution was plated on plate count agar for bacterial enumeration. In case of unwashed #1382 fabric, very few bacteria were attached to the surface (60 ± 72 cfu for E. coli and 6 ± 11 cfu for Staphylococcus aureus) and after washing of the surface, there was no bacterial recovery. This indicates that the surface has less bacterial attachment, and if washed the attached bacteria can easily be removed. On the other hand, coated #1386 fabric did not show any bacterial attachment to the surface for both the pathogens compared to uncoated yellow fabric 1384 (9.3 × 103 cfu for E. coli and 4 × 102 cfu for Staphylococcus aureus). The droplet containing bacteria wetted the aluminum sheet surface of 1381 and 1434 samples, and the bacteria were recovered from both the surfaces even after washing three times. On the other hand, the drop of solution containing bacteria beaded up on the surface of aluminum sample 1437 exhibiting superhydrophobic property and showed no bacterial recovery for both the pathogens. Our results thus clearly indicate that surface-modified fabrics (1382 and 1386) and aluminum sheet (1437) do not allow bacterial attachment and therefore could be suitable for their application in developing self-cleaning aircraft and cabin crew surfaces materials.

Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect

The antimicrobial activity of silver-coated carbon nanotubes (AgCNTs) and their potential mode of action against mucoid and nonmucoid strains of Pseudomonas aeruginosa was investigated in vitro. The results showed that AgCNTs exhibited antimicrobial activity against both strains with minimum inhibitory concentrations of approximately 8 µg/mL, indicating a high sensitivity of P. aeruginosa to AgCNTs. AgCNTs were also bactericidal against both strains at the same minimum inhibitory concentration. Scanning and transmission electron-microscopy studies further revealed that a majority of the cells treated with AgCNTs transformed from smooth rod-shape morphology to disintegrated cells with broken/damaged membranes, resulting in leakage of cytoplasmic contents to produce ghost cells. The molecular effects of AgCNTs on P. aeruginosa genes involved in virulence and pathogenicity, stress response, and efflux pumps were evaluated for changes in their expression. Quantitative real-time PCR (qRT-PCR) showed that after exposure to AgCNTs, the expression levels of the rpoS, rsmZ, and oprD genes were significantly downregulated in both strains of P. aeruginosa compared to the untreated samples. These results suggest that the mechanism of action of AgCNTs may be attributed to their effect on cell-membrane integrity, downregulation of virulence-gene expression, and induction of general and oxidative stress in P. aeruginosa.

Novel cationic peptide TP359 down-regulates the expression of outer membrane biogenesis genes in Pseudomonas aeruginosa: a potential TP359 anti-microbial mechanism

BackgroundAntimicrobial peptides (AMPs) are a class of antimicrobial agents with broad-spectrum activities. Several reports indicate that cationic AMPs bind to the negatively charged bacterial membrane causing membrane depolarization and damage. However, membrane depolarization and damage may be insufficient to elicit cell death, thereby suggesting that other mechanism(s) of action could be involved in this phenomenon. In this study, we investigated the antimicrobial activity of a novel antimicrobial peptide, TP359, against two strains of Pseudomonas aeruginosa, as well as its possible mechanisms of action.ResultsTP359 proved to be bactericidal against P. aeruginosa as confirmed by the reduced bacteria counts, membrane damage and cytoplasmic membrane depolarization. In addition, it was non-toxic to mouse J774 macrophages and human lung A549 epithelial cells. Electron microscopy analysis showed TP359 bactericidal effects by structural changes of the bacteria from viable rod-shaped cells to those with cell membrane damages, proceeding into the efflux of cytoplasmic contents and emergence of ghost cells. Gene expression analysis on the effects of TP359 on outer membrane biogenesis genes underscored marked down-regulation, particularly of oprF, which encodes a major structural and outer membrane porin (OprF) in both strains studied, indicating that the peptide may cause deregulation of outer membrane genes and reduced structural stability which could lead to cell death.ConclusionOur data shows that TP359 has potent antimicrobial activity against P aeruginosa. The correlation between membrane damage, depolarization and reduced expression of outer membrane biogenesis genes, particularly oprF may suggest the bactericidal mechanism of action of the TP359 peptide.

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium

BackgroundSynthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes.ResultsMeasurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction.ConclusionsThis study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Anti-RSV peptide-loaded liposomes for the inhibition of respiratory syncytial virus

Although respiratory syncytial virus (RSV) is one of the leading causes of acute respiratory tract infection in infants and adults, effective treatment options remain limited. To circumvent this issue, there is a novel approach, namely, the development of multifunctional liposomes for the delivery of anti RSV-peptides. While most of the peptides that are used for loading with the particulate delivery systems are the penetrating peptides, an alternative approach is the development of liposome-peptide systems, which are loaded with an RSV fusion peptide (RF-482), which has been designed to inhibit the RSV fusion and block infection. The results of this work have revealed that the liposomes themselves can serve as potential RSV inhibitors, whilst the anti-RSV-peptide with liposomes can significantly increase the RSV inhibition when compared with the anti-RSV peptide alone.

106 ASSESSMENT OF ANTI-BACTERIAL EFFECTS OF PEGYLATED SILVER-COATED CARBON NANOTUBES ON CAUSATIVE BACTERIA OF BOVINE INFERTILITY USING BIOLUMINESCENCE IMAGING SYSTEM

Pathogenic bacteria including Escherichia coli and Salmonella sp. are the major causative agents of endometritis and can cause infertility in livestock animals. Antibiotics are commonly used to terminate bacterial infections, but the development of bacterial antibiotic resistance is often encountered. Nanotechnology associated with silver nanoparticles has been highlighted as an alternative anti-bacterial agent, and pegylated silver-coated single-walled carbon nanotubes have high anti-bacterial effects and are non-toxic to human and murine cells in vitro. Here we verified whether a real-time bioluminescence monitoring system could be an alternative tool to assess anti-bacterial effects of nanotubes in a noninvasive approach. Escherichia coli and Salmonella sp. were transfected with plasmids containing constructs for luciferase enzyme (LuxCDABE) and substrate (luciferin) to create self-illuminating bioluminescent bacteria. Pathogens were grown in LB broth at 37°C, adjusted to 107 cfumL-1, and placed in 96-well plates for treatments. Pegylated (pSWCNTs-Ag) and non-pegylated (SWCNTs-Ag) nanotubes were prepared and added to culture wells at various concentrations (31.25-125µgmL-1). The control group corresponded to bacteria without nanotubes (0µgmL-1). Anti-bacterial effects of nanotubes were determined every 10min until 1h, then every 30min up to 6h incubation through optical density (600nm) measurements and bioluminescence imaging (BLI) and quantification using an IVIS system. Optical density and BLI data were compared at each time-point using 2-way ANOVA, with P<0.05 set for significance. Bioluminescence signals emitted by both bacteria stains appeared within 10min of incubation. Thereafter, control bacteria showed exponential growth that was detected as early as 25min post-incubation. Bioluminescence imaging revealed dose-dependent anti-bacterial activities of both pSWCNTs-Ag and SWCNTs-Ag on each E. coli and Salmonella sp. (P<0.05). Contrary to BLI, the OD values did not always reflect bacteria concentrations, and varied according to nanotube concentrations. No significant differences in anti-bacterial activities were revealed between pSWCNTs-Ag and SWCNTs-Ag based on OD values during 6h of incubation (P>0.05); meanwhile, pSWCNTs-Ag nanotubes exhibited stronger anti-bacterial effects than SWCNTs-Ag during the same period using BLI (P<0.05). In summary, we confirmed previous reports showing dose-dependent eliminations of pathogenic bacteria by silver nanotubes. Pegylated nanotubes exhibited high anti-bacterial activity compared to non-pegylated nanotubes. Bioluminescence imaging system revealed superior resolution to enable precise investigation of anti-bacterial kinetics of silver nanotubes. This feature could be useful for the study of bacterial infections that impair livestock fertility.