Modification, crosslinking and reactive electrospinning of a thermoplastic medical polyurethane for vascular graft applications.

Thermoplastic polyurethanes are used in a variety of medical devices and experimental tissue engineering scaffolds. Despite advances in polymer composition to improve their stability, the correct balance between chemical and mechanical properties is not always achieved. A model compound (MC) simulating the structure of a widely used medical polyurethane (Pellethane) was synthesized and reacted with aliphatic and olefinic acyl chlorides to study the reaction site and conditions. After adopting the conditions to the olefinic modification of Pellethane, processing into flat sheets, and crosslinking by thermal initiation or ultraviolet radiation, mechanical properties were determined. The modified polyurethane was additionally electrospun under ultraviolet light to produce a crosslinked tubular vascular graft prototype. Model compound studies showed reaction at the carbamide nitrogen, and the modification of Pellethane with pentenoyl chloride could be accurately controlled to up to 20% (correlation: rho=0.99). Successful crosslinking was confirmed by insolubility of the materials. Initiator concentrations were optimized and the crosslink densities shown to increase with increasing modification. Crosslinking of Pellethane containing an increasing number of pentenoyl groups resulted in decreases (up to 42%, p<0.01) in the hysteresis and 44% in creep (p<0.05), and in a significant improvement in degradation resistance in vitro. Modified Pellethane was successfully electrospun into tubular grafts and crosslinked using UV irradiation during and after spinning to render them insoluble. Prototype grafts had sufficient burst pressure (>550 mm Hg), and compliances of 12.1+/-0.8 and 6.2+/-0.3%/100 mm Hg for uncrosslinked and crosslinked samples, respectively. It is concluded that the viscoelastic properties of a standard thermoplastic polyurethane can be improved by modification and subsequent crosslinking, and that the modified material may be electrospun and initiated to yield crosslinked scaffolds. Such materials hold promise for the production of vascular and other porous scaffolds, where decreased hysteresis and creep may be required to prevent aneurismal dilation.

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium.

Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.