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Development of a new biomaterial associated with human keratinocytes for use as a skin substitute
The replacement of large damaged skin areas, such as those resulting from major trauma, burns and skin cancer is an old challenge for medicine. Autografts are limited to the donor site and xenografts and allografts are of limited use due to tissue immune reactions and the possibility of diseases transmission. Since the currently available treatments are insufficient to prevent scar formation and promote complete tissue regeneration, tissue engineering is an alternative for the development of a more efficient skin substitute. With the idea of encountering such a skin substitute, the aim of the current study has been to produce an EGF enriched biomaterial using PDLLA polymer and Type 1 Collagen for use as a scaffold for tissue engineered skin substitutes.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
For this proposal, scaffolds were constructed by the coaxial electrospinning technique and divided into 3 groups: 1) PDLLA – a coaxial PDLLA fiber, 2) PDLLA/EGF – a coaxial fiber with EGF/albumin solution core and 3) PDLLA/Collagen - a PDLLA/EGF scaffold with Type 1 collagen coating. Immortalized human keratinocytes (HaCaT) were seeded on top of the scaffolds for biological analysis. Random fibers were obtained for all the groups without beads, and characterization of the scaffolds was achieved by scanning electron microscopy (SEM) for morphology and pore size evaluation. Confocal microscopy was used to visualize core-shell structure and Fourier transform infrared spectroscopy (FTIR) to analyze the presence of collagen on the fibers. Contact angle measurements (WCA) were performed for hydrophilicity measurements. The fiber diameters for group 1 was 1.293±0.320 µm, 1.235±0.486 µm for group 2 and 1,219±0.423 µm for group 3. All the groups showed similar pore size, being 7.750 µm, 7.410 µm and 7.314 µm for groups 1, 2 and 3, respectively. Core-shell relation was confirmed by confocal microscopy and FTIR analysis showed a strong peak in 1,540 - 1,660 cm-1 region in group 3. This was probably due to stretching vibrations of the peptide C=O groups (Amid I), which suggests the presence of collagen in these fibers. Furthermore, the WCA for group 3 was 108.69º while for group 1 it was 118.21º and 116.58º for group 2. The groups were evaluated for cell viability and cytotoxicity on days 1, 7 and 14. As a result, on day 1, cell viability was greater in the PDLLA/Collagen scaffolds with an absorbance of 0.622±0.059 in comparison with 0.349±0.063 for the control (PDLLA scaffold) and 0.507±0.102 for the PDLLA/EGF scaffold. On day 7, the absorbance for the PDLLA scaffold was 0.486±0.140, the PDLLA/EGF group was 0.616±0.004 and the PDLLA/Collagen was 0.793±0.175. On day 14, absorbance for groups 1, 2 and 3 were 0.474±0.081, 0.442±0.020 and 0.410±0.030, respectively. In terms of the biological analysis, the PDLLA/Collagen group showed the best results for cell viability tests up to day 7, with no significant difference on day 14. Cytotoxicity assays through LDH measurements performed on days 1, 7 and 14 showed statistical significance (p<0.05) when comparing the PDLLA group with the PDLLA/EGF and PDLLA/Collagen groups on day 14.
Considerações Finais/Final considerations/Consideraciones finales
In conclusion, the PDLLA/EGF and mainly the PDLLA/Collagen groups, showed good results for cell growth, with the presence of viable cells. New in vitro studies are currently being performed to evaluate the full potential of this new biomaterial, which could become an option for skin substitute studies.
Palavras-chave/Key words/Palabras clave
skin substitute, EGF, collagen, keratinocytes
BRUNO JC ALCANTARA, Daniela Steffens, Patricia Pranke