Dados do Trabalho

Título/Title/Titulo

Recombinant Human Growth Factors and Mesenchymal Stem Cells derived Exosomes for Wound Healing and Skin Regeneration

Introdução/Introduction/Introdución

The regenerative medicine aims treatments to accelerate the different phases of tissue repair: blood clotting and inflammation, cell proliferation, tissue remodeling. In scarring and deficient tissues, there is an impairment of the ideal levels of growth factors in the wound healing phases. In chronic wounds, protease levels exceed that of their respective inhibitors, leading to destruction of extracellular matrix and degradation of growth factors and their receptors. By manipulating the growth factors composition, it is possible to accelerate or modify the process of regeneration and remodeling of damaged tissues. Currently, Mesenchymal Stem Cells (MSCs) are considered to be promising sources for various types of Cell Therapies in the Regenerative Medicine field. These cells display a regenerative potential and may be used for repair and maintenance of different tissue types, however, the mechanisms by which this repair activity is exerted is not well known yet. In this way, the application of MSCs or their microvesicles/exosomes and growth factors could contribute to the correct repair in a timely manner.

Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados

First, this study aims to establish a model of dorsal wound healing in nude rats in order to test in vivo biological activity of recombinant human Platelet-derived growth factor (rhPDGF-BB) and Vascular endothelial growth factor (rhVEGF165), abundant factors at Platelet-rich plasm (PRP), but produced in mammalian cells, and, further, use this model to evaluate the role of MSCs and their exosomes, combined with recombinant growth factors, to verify its therapeutic effect on the tissue regeneration. The model of dorsal wound healing was standardized in nude rowett rats using four wounds with surgical punch of 6 mm diameter. rhPDGF-BB and rhVEGF165 were purified using heparin affinity chromatography, quantified by ELISA and 5ug were applied into each injury. The wounds were photodocumented and after seven days the wound skin was collected for histological analysis with Hematoxylin-Eosin, Picro-Sirius and Masson's Trichrome to evaluate the new tissue formed. The results showed comparative efficacies in both treatments of wounds with rhPDGF-BB or rhVEGF165, with an accelerate cicatrization rate in comparison with the injury without treatment or treated with only the vehicle (saline solution). To improve the cicatrization, for the next step we isolated exosomes from MSCs. We standardized a protocol with three steps: ultrafiltration (10kDa cut), Total Exosome Isolation Reagent kit and ultracentrifugation at 120.000g. The material isolated was evaluated by Western blot assay using CD81 antibody and the presence of exosomes was detected at 26kDa. This material will be applied combined with recombinant growth factors, described before, at injuries to evaluate its wound potential.

Considerações Finais/Final considerations/Consideraciones finales

We waiting to develop and offer an improved alternative and approach to wound healing treatment for patients and animals with complex chronic ulcers and other damage in the skin, which are difficult to treat and associated with high treatment costs.
Ethical approval: CEUA-FMVZ/USP nº 8941120916
Funding support: CNPq, CAPES, BNDES, FINEP, MS-DECIT, MCTI.

Palavras-chave/Key words/Palabras clave

wound healing, PDGF-BB, VEGF, mesenchymal stem cells, exosome.