Martin Pradny

Treatment of Ocular Surface Injuries by Limbal and Mesenchymal Stem Cells Growing on Nanofiber Scaffolds

Stem cell (SC) therapy represents a promising approach to treat a wide variety of injuries, inherited diseases, or acquired SC deficiencies. One of the major problems associated with SC therapy remains the absence of a suitable matrix for SC growth and transfer. We describe here the growth and metabolic characteristics of mouse limbal stem cells (LSCs) and mesenchymal stem cells (MSCs) growing on 3D nanofiber scaffolds fabricated from polyamide 6/12 (PA6/12). The nanofibers were prepared by the original needleless electrospun Nanospider technology, which enables to create nanofibers of defined diameter, porosity, and a basis weight. Copolymer PA6/12 was selected on the basis of the stability of its nanofibers in aqueous solutions, its biocompatibility, and its superior properties as a matrix for the growth of LSCs, MSCs, and corneal epithelial and endothelial cell lines. The morphology, growth properties, and viability of cells grown on PA6/12 nanofibers were comparable with those grown on plastic. LSCs labeled with the fluorescent dye PKH26 and grown on PA6/12 nanofibers were transferred onto the damaged ocular surface, where their seeding and survival were monitored. Cotransfer of LSCs with MSCs, which have immunosuppressive properties, significantly inhibited local inflammatory reactions and supported the healing process. The results thus show that nanofibers prepared from copolymer PA6/12 represent a convenient scaffold for growth of LSCs and MSCs and transfer to treat SC deficiencies and various ocular surface injuries.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat

OBJECT Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

Biopolymer-based degradable nanofibres from renewable resources produced by freeze-drying

We describe a new biopolymer-based nanofibrous material possibly suitable for tissue engineering prepared by an environment-friendly organic solvent-free method. Glycogen, a biodegradable hyperbranched D-glucose polymer, comes from renewable resources and is normally present in man. It forms nanofibres by simple freeze-drying from aqueous solutions with concentration less than 0.5%. However, the architecture of the freeze-dried material depends on the starting biopolymer concentration within the tested range 0.1–5 wt%; in particular higher concentrations produce porous sponge-like structures with communicating pores. Because of the solubility of glycogen in water, nanofibres were modified by solvent-free grafting biodegradable poly(ethyl cyanoacrylate) from vapor phase. Exposing glycogen nanofibres to vapors of ethyl cyanoacrylate only slightly changed the material architecture while producing a water-insoluble biodegradable material with glycogen-to-poly(ethyl cyanoacrylate) ratio depending on the polymerization time. The material was proven to be hydrolytically degradable over the course of several months.

Hydrogels Contact Lenses

Contact lenses can be classified in a number of ways; however, the two main categories are hard and soft lenses, which are based on the material used for their manufacture. The soft lens category can be further divided into hydrophobic and hydrophilic subcategories. Consequently, the development of contact lens materials took three specific directions: hydrogels with high water content, rigid gas-permeable lenses with enhanced oxygen permeability, and surface modification of silicone elastomer lenses. These polymeric systems are expected to improve the water content of the contact lenses as well as the permeability to oxygen, which are crucial properties but controllable through the molecular design. Currently, the high water content hydrogels are being challenged by the silicone-hydrogels for the world market share.

Bioactive support for cell cultivation and potential grafting. Part 1: Surface modification of 2-hydroxyethyl methacrylate hydrogels for avidin immobilization

Abstract Synthetic hydrogels are often used in biomedical applications as many of them are compatible with living tissue and moreover they can meet most criteria for artificial tissue properties. For applications in tissue engineering modification of polymer surface using some bioactive compounds (e.g. saccharides, proteins) for promoting the process of cell adhesion and proliferation is widely used. In this work, a series of modified hydrogels was prepared by three different methods: by copolymerization of 2-hydroxyethyl methacrylate with methacrylic acid, by hydrolysis and oxidation of poly(2-hydroxyethyl methacrylate) to obtain carboxyl-rich supports. The influence of the reaction conditions of the hydrogel surface treatment on the total carboxylic group content and the swelling degree was studied. The modified hydrogels were characterized by attenuated total reflectance FT-IR spectroscopy and refractive index measurements. Obtained carboxylic groups on the hydrogel surface have allowed the immobilization of avidin in two ways: 1) electrostatically through dissociated carboxylic groups and 2) covalently bonded through activated carboxylic groups by Nhydroxysuccinimide. The capacity of hydrogels for avidin immobilization was determined by Bradford spectrophotometric method. The results so far obtained from the preliminary biological tests showed that immobilized avidin on the hydrogel surface provides better adhesion and proliferation of keratinocytes compared to supports without avidin.

Biphasic Equilibrium Dialysis of Poly(N-Isopropyl Acrylamide) Nanogels Synthesized at Decreased Temperatures for Targeted Delivery of Thermosensitive Bioactives

Hydrogel nanoparticles, referred to also as nanogels, are of special interest for medical and pharmaceutical applications. Due to small size in the range below the diameter of the capillaries, they are proposed as drug delivery carriers. The aim of the study was to estimate the influence of composition and reaction conditions during synthesis of poly-N-isopropyl acrylamide cross-linked by polyethylene glycol diacrylate on the purification rates of the polymer. Six types of thermosensitive nanogels were prepared by surfactant-free dispersion polymerization and assessed in terms of process yield, composition, and size at temperatures below and over volume phase temperature. During the diffusion of impurities, in the course of dialysis, assessed by the conductometric method, the remarkable influence of temperature and initiator concentration on the process was revealed. The release rates varied in the range between 9.63 · 10−2 and 1.39 · 10−1 h−1 in the first stage of the process, whereas in the second stage they were between 2.09 · 10−2 and 6.28 · 10−2 h−1. The evaluated time to obtain acceptable purity of the preparation was estimated to be in the range of 18 days. More detailed research should be directed towards the influence of the structure of obtained material on the purification process.

Morphological and chromatographic characterization of molecularly imprinted monolithic columns.

Abstract The chromatographic stationary phases based on molecularly imprinted monoliths were prepared by free radical polymerization and subsequently characterized. The mixture of commonly used comonomers of ethylene dimethacrylate/methacrylic acid was polymerized in presence of various porogenic mixtures. Tosyl-L-phenylalanine was selected as a model template. Polymerization steps were carried out in the glass columns (i.d. of 3.3 mm) enabling UV initiation. Difficulties encountered with polymerization in situ, e.g. volume contraction and adhesion of polymer onto the glass inner wall, were successfully solved. The morphology of monoliths was investigated by electron microscopy, mercury porosimetry and surface area measurements. The influence of polymerization conditions on monolith morphology and subsequent chromatographic properties was evaluated. Polymers prepared by UV polymerization showed higher total porosity and the most frequent pore radius compared to the same polymers prepared thermally. The prepared monoliths by UV irradiation were also significantly more permeable for mobile phase during the chromatography tests. Morphology of monoliths prepared in dodecanol/toluene porogenic mixture markedly depended on the temperature of polymerization; the most frequent pore radius decreased with increasing temperature. From chromatographic point of view, monolithic columns were tested by separations of standard hydrophobic solutes. Height equivalent to a theoretical plate reached the values of approx. 25 μm, Walters indices of hydrophobicity laid slightly above 3. Prepared monoliths were compared to the conventional columns, filled with the particles prepared from the polymer of the same composition and preparation. In many cases the monolithic columns showed better chromatographic parameters.