No abstract

In the regeneration process for new tissues, oxygen promotes re-epithelialization and healing of infected wounds, increases keratinocyte differentiation, proliferation and migration of fibroblast, and induces angiogenesis, collagen synthesis and wound contraction. Therefore, provision of oxygen to cells and tissues at an optimal level is critical for effective tissue regeneration and wound healing. In this study, we developed sustained oxygen-releasing polymeric microspheres and fabricated a sponge type dressing by embedding the microspheres into alginate-based hydrogel that can supply oxygen to wounds. We further investigated the applicability of the microspheres and hydrogel sponge to wound healing in vitro and in vivo. Oxygen-releasing microspheres (ORM) were made by incorporating hydrogen peroxide (H2O2) into poly(lactic-co-glycolic acid) (PLGA) using double emulsion method. H2O2-PLGA microspheres were embedded into alginate-based hydrogel to form a porous oxygen-releasing hydrogel sponge (ORHS). Biocompatibility was performed using cell counting kit-8. The oxygen release kinetic study was performed using a hydrogen peroxide assay kit and oxygen meter. The wound healing potential of ORHS was evaluated using the wound scratch model. In vivo studies were carried out to investigate the safety and efficacy of the ORHS for wound healing. Experimental results confirmed that oxygen released from ORMand ORHS induced neovascularization and promoted cell proliferation thereby facilitating effective wound healing. It is suggested that the ORM can be used for supplying oxygen to where cells and tissues are deprived of necessary oxygen, and ORHS is an intelligent scaffold to effectively heal wound by enhanced angiogenesis by oxygen. Conclusively, oxygen releasing polymeric microspheres and hydrogel scaffolds have potential for a variety of tissue engineering applications, where require oxygen.

Recent studies have revealed the potential of B-type natriuretic peptide (BNP) as a good prognostic marker for patients with heart failure. Antibodies against BNP are expected to be usefully employed in the diagnosis of heart failures. We established a more efficient method to produce functional anti-BNP, single chain variable fragment (scFv) using a eukaryotic expression system of Pichia pastoris. Although analysis of the N-terminal (NT) sequence of the expressed anti-BNP scFv indicated that the two Ste13 sites of the secreted anti-BNP scFv were not cleaved, the specificity of anti-BNP scFv was not affected significantly. The binding activity of anti-BNP scFv against other antigens, against four other antigens, NT probrain peptide (NT-pro BNP), atrial natriuretic peptide (ANP), carcinoembryonic antigen (CEA) and human serum albumin (HSA), was only one thousandth that of the original BNP antigen, which clearly demonstrated the specific binding of recombinant scFv toward BNP. The anti-BNP, scFv-based, electrochemical immunoassay exhibited excellent analytical performance with a detection limit of 1 fg/ml and a wide linear detection range from 1 to 10,000 fg/ml. The optimum culture conditions to obtain the maximum concentration of recombinant scFv were a pH range of 5.0-7.0 and an incubation temperature of 20°C. This anti-BNP scFv expressed in P. pastoris has the potential for promising applications in the diagnosis of heart failure.