ABSTRACT
Background: Reconstruction of large eyelid defects remains challenging due to the lack of suitable eyelid tarsus tissue substitutes. We aimed to evaluate a novel bioengineered chitosan scaffold for use as an eyelid tarsus substitute. Methods: Three-dimensional macroporous chitosan hydrogel scaffold were produced via cryogelation with specific biomechanical properties designed to directly match characteristics of native eyelid tarsus tissue. Scaffolds were characterized by confocal microscopy and tensile mechanical testing. To optimise biocompatibility, human eyelid skin fibroblasts were cultured from biopsy-sized samples of fresh eyelid skin. Immunological and gene expression analysis including specific fibroblast-specific markers were used to determine the rate of fibroblast de-differentiation in vitro and characterize cells cultured. Eyelid skin fibroblasts were then cultured over the chitosan scaffolds and the resultant adhesion and growth of cells were characterized using immunocytochemical staining. Results: The chitosan scaffolds were shown to support the attachment and proliferation of NIH 3T3 mouse fibroblasts and human orbital skin fibroblasts in vitro. Our novel bioengineered chitosan scaffold has demonstrated biomechanical compatibility and has the ability to support human eyelid skin fibroblast growth and proliferation. Conclusions: This bioengineered tissue has the potential to be used as a tarsus substitute during eyelid reconstruction, offering the opportunity to pre-seed the patient's own cells and represents a truly personalised approach to tissue engineering.
Subject(s)
Chitosan/chemistry , Eyelids/physiology , Tissue Engineering , Tissue Scaffolds/chemistry , Animals , Cell Line , Cell Proliferation , Eyelids/pathology , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Hydrogels/chemistry , Mice , Porosity , Regeneration , Tensile Strength , Tubulin/metabolismABSTRACT
Dermal defects caused by trauma or disease are challenging to treat due to difficult-to-treat infections that impair wound healing. Due to the widespread emergence of drug-resistant bacteria and dwindling discoveries of new antibiotics, there is currently an urgent need to introduce novel antimicrobials effective against antibiotic-resistant bacteria without causing damage to host tissues. As selenium (Se) and silver (Ag) are known for their antimicrobial properties, we investigated the separate loading of these materials into porous chitosan/PVA (CS) scaffolds through a simple in situ deposition method to create two distinct wound dressing materials (CS-Se and CS-Ag). Scaffolds with Se nanostructures and scaffolds containing Ag nanostructures were characterized and their activities against S. aureus - (a Gram-positive bacterium), E. coli - (a Gram-negative bacterium) and Methicillin-Resistant S. aureus (MRSA) - (a multi-drug resistant bacterium) were compared. The release of Ag and Se in vitro was shown to depend strongly on the release medium used (deionised water, mammalian or bacterial culture media). Ag-loaded scaffolds showed a significant reduction in CFUs and cytotoxicity towards fibroblasts while Se-loaded scaffolds showed abilities to damage bacterial cell membrane and non-toxicity to fibroblast. Overall, in this study we have demonstrated simple, in situ immobilization porous CS scaffolds with either Se or Ag nanostructures which could be used to suit different wound healing applications.
ABSTRACT
Antimicrobial agents that have no or low cytotoxicity and high specificity are desirable to have no or minimal side effects. We report here the low cytotoxicity of polyvinyl alcohol-stabilized selenium (Se) nanoparticles and their differential effects on growth of S. aureus, a gram-positive bacterium and E. coli, a gram-negative bacterium. The nanoparticles were synthesised through redox reactions in an aqueous environment at room temperature and were characterised using UV visible spectrophotometry, transmission electron microscopy, dynamic light scattering and x-ray photoelectron spectroscopy. The nanoparticles showed low toxicity toward fibroblasts which remained more than 70% viable at Se concentrations as high as 128 ppm. The nanoparticles also exhibited very low haemolysis with only 18% of maximal lysis observed at a Se concentration of 128 ppm. Importantly, the nanoparticles showed strong growth inhibition toward S. aureus at a concentration as low as 1 ppm. Interestingly, growth of E. coli was unaffected at all concentrations tested. This study therefore strongly suggests that these nanoparticles should be investigated further to understand this differential effect as well as for potential advanced antimicrobial applications such as S. aureus infection-resisting, non-cytotoxic coatings for medical devices.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Survival/drug effects , Escherichia coli/drug effects , Selenium/chemistry , Selenium/pharmacology , Staphylococcus aureus/drug effects , 3T3 Cells , Animals , Anti-Bacterial Agents/toxicity , Escherichia coli Infections/prevention & control , Fibroblasts/drug effects , Hemolysis/drug effects , Horses , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Mice , Microbial Sensitivity Tests , Selenium/toxicity , Staphylococcal Infections/prevention & controlABSTRACT
Nano-/micrometer-scaled films and capsules made of low-fouling materials such as poly(ethylene glycol) (PEG) are of interest for drug delivery and tissue engineering applications. Herein, the assembly and degradation of low-fouling, alkyne-functionalized PEG (PEG(Alk) ) multilayer films and capsules, which are prepared by combining layer-by-layer (LbL) assembly and click chemistry, are reported. A nonlinear, temperature-responsive PEG(Alk) is synthesized, and is then used to form hydrogen-bonded multilayers with poly(methacrylic acid) (PMA) at pH 5. The thermoresponsive behavior of PEG(Alk) is exploited to tailor film buildup by adjusting the assembly conditions. Using alkyne-azide click chemistry, PEG(Alk)/PMA multilayers are crosslinked with a bisazide linker that contains a disulfide bond, rendering these films and capsules redox-responsive. At pH 7, by disrupting the hydrogen bonding between the polymers, PEG(Alk) LbL films and PEG(Alk) -based capsules are obtained. These films exhibit specific deconstruction properties under simulated intracellular reducing conditions, but remain stable at physiological pH, suggesting potential applications in controlled drug release. The low-fouling properties of the PEG films are confirmed by incubation with human serum and a blood clot. Additionally, these capsules showed negligible toxicity to human cells.