Utilização do polímero natural polihidroxibutirato-cohidroxivalerato para auxiliar a vascularização e regeneração da pele a partir de princípios da engenharia de tecidos

Abstract

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules to induce tissue regeneration. Despite its enormous potential and current achievements, up to this day, only products developed for skin and cartilage are available for clinical applications. However, these products still face some limitations. The use of allogenic compounds and also the lack of vascularization undermine the effectiveness of skin regeneration within these products. In this sense, the first part of this work aimed to promote endothelial differentiation from adiposederived stem cells (ASC) seeded on electrospun fiber mesh made of poly(3hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV or PHB-HV) in the ratio 30:70 respectively. Using the electrospinning technique it was possible to obtain membranes formed by fibers with diameters between 300nm and 1.3μm. The morphology of these membranes increased adhesion and proliferation of ASCs. When induced to endothelial differentiation, the cells expressed specific markers and reorganized forming capillary-like structures. In the second stage, PHBV bilayer structures that mimic the physical structure of skin were designed and produced by assembling a solvent cast membrane and a 3D freeze dried scaffold. The combination of the distinct qualities of the two systems such as susceptibility to enzymatic degradation, high water retention capability and stiff character, allowed achieving an improved system expected to contribute to a better wound healing. The bilayer structures design and associated properties favored human fibroblasts and keratinocytes performance under defined heterotypic culture conditions forming biologically interactive dermal and epidermal analogs that lead to the particular rearrangement of keratinocytes into multiple layers resembling epidermis-like organization. These results demonstrate that the proposed PHBV-based bilayer structure has potential to be used as an improved skin graft. Therefore, the last step of this thesis was to evaluate in vivo the association of the angiogenic properties of ASCs and functional morphology of PHBV bilayer structure to promote better tissue regeneration of the skin using an excision rat model. From the histological analysis and gene transcripts quantification it was possible to determine that the PHBV bilayerstructure has suitable properties to promote skin regeneration with lower propensitiy to form scar tissue. Moreover, the association with ASCs provided an even more favorable environment that leaded to the formation of a new tissue more similar to the normal skin. In general, our results demonstrate that PHBV is a potential polymer to be used in the development of new scaffolds based on different processing techniques that can favor the vascularization and skin tissue regeneration.