No abstract

We report on the design and fabrication of a frame-supported nanofibrous membrane for the transplantation of retinal pigment epithelial (RPE) cells, which is a promising therapeutic option for the treatment of degenerative retinal disorders. The membranous cell carrier prepared from 640 nm-thick poly(DL-lactide) fibres uniquely combines high porosity, large pore size and low thickness, to maximize the nutrient supply to the transplanted cells in the subretinal space and thus to enhance the therapeutic effect of the transplantation. The carrier was prepared by electrospinning, which made it easy to embed a 95 μm-thick circular supporting frame 2 mm in diameter. Implantations into enucleated porcine eyes showed that the frame enabled the ultrathin membrane to be handled without irreversible folding, and allowed the membrane to regain its flat shape when inserted into the subretinal space. We further demonstrated that the minimum membrane thickness compatible with the surgical procedure and instrumentation employed here was as low as 4 μm. Primary porcine RPE cells cultivated on the membranes formed a confluent monolayer, expressed RPE-specific differentiation markers and showed transepithelial resistance close to that of the native RPE. Most importantly, the majority of the RPE cells transplanted into the subretinal space remained viable. The ultrathin, highly porous, and surgically convenient cell carrier presented here has the potential to improve the integration and the functionality of transplanted RPE cells.

We present an investigation of the preparation of highly porous hydrogels based on biodegradable synthetic poly(α-amino acid) as potential tissue engineering scaffolds. Covalently cross-linked gels with permanent pores were formed under cryogenic conditions by free-radical copolymerization of poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-N(5)-(2-methacryloyl-oxy-ethyl)-L-glutamine] (PHEG-MA) with 2-hydrohyethyl methacrylate (HEMA) and, optionally, N-propargyl acrylamide (PrAAm) as minor comonomers. The morphology of the cryogels showed interconnected polyhedral or laminar pores. The volume content of communicating water-filled pores was >90%. The storage moduli of the swollen cryogels were in the range of 1-6 kPa, even when the water content was >95%. The enzymatic degradation of a cryogel corresponded to the decrease in its storage modulus during incubation with papain, a model enzyme with specificity analogous to wound-healing enzymes. It was shown that cryogels with incorporated alkyne groups can easily be modified with short synthetic peptides using azide-alkyne cycloaddition "click" chemistry, thus providing porous hydrogel scaffolds with biomimetic features.

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell-adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co-polymerization of methacryloylated macromonomer poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-L-alanine-stat-methacryloyllysine] with 2-hydroxyethyl methacrylate (HEMA) as minor co-monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl-GGGRGDSG-OH and methacryloyl-GGGYIGSR-OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5-20 microm), dependent on the polyHEMA phase separation in water, was followed by low-vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells.

The technique for fabrication of soft porous hydrogels, in which both the size and the orientation of inner pores can be controlled, was developed. Three-dimensional hydrophilic gels based on poly[2-hydroxyethyl methacrylate] are designed as scaffolds for regeneration of soft tissues, e.g., nerve tissue. Anisotropic macropores of the size ranging from 10 to 50 microm were formed (1) by using a porogen-leaching method with a solid organic porogen, (2) by phase-separation during gelation in solvent-nonsolvent mixture, or (3) by combination of solid porogen elimination and phase-separation. As a porogen, poly(L-lactide) fibers were applied and consequently washed away under mild conditions to obtain desired spatial orientation of pores. Highly water-swollen polymer gels were characterized with high pressure (low vacuum) scanning electron microscopy (AquaSEM). The morphology of voids remaining after removing the solid PLLA porogen (the macropores) was clearly shown.