Efeito do laser de baixa intensidade na atividade biológica de células-tronco do ligamento periodontal humano cultivadas sobre arcabouços de quitosana

• Ano de defesa: 2019
• Autor(a) principal: Cavalcanti, Rafaella Bastos Leite
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Brasil; UFRN; PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS ODONTOLÓGICAS
• Palavras-chave em Português: Células-tronco mesenquimais; Ligamento periodontal; Biomateriais; Quitosana; Irradiação a laser de baixa intensidade; Proliferação celular; CNPQ::CIENCIAS DA SAUDE::ODONTOLOGIA
• Link de acesso: https://repositorio.ufrn.br/jspui/handle/123456789/27757

Low-level laser irradiation (LLLI) is able to stimulate the proliferation of various cell types, but little is known about its effectiveness on the proliferation of cells cultured on biomaterial surfaces. The aim of this study was to evaluate the influence of LLLI on the proliferation and viability of human periodontal ligament stem cells (PDLSCs) cultured on chitosan scaffolds. Chitosan was submitted to tests to identify the real mass content and degree of deacetylation. Chitosan membranes were prepared by solvent evaporation technique and submitted to morphology and surface characterization. PDLSCs previously isolated and characterized were grown on the surfaces of four groups: (P) culture plate plastic, non-irradiated, as a positive control of cell growth; (C) chitosan, non-irradiated; (L1) chitosan irradiated with a dose of 1 J/cm²; and (L4) chitosan, irradiated with 4 J/cm². The irradiations were performed with InGaAlP diode laser with wavelength of 660 nm, power 30 mW, tip diameter of 0.01cm², and continuous action mode in a single dose. Cell viability and proliferation were evaluated by Alamar Blue, Live/Dead, Annexin V/PI and Ki67 assays, as well as cell cycle analysis, whereas cell morphology was evaluated by MEV. The data showed that the chitosan presented a real mass content of 88.08% and degree of deacetylation of 91.37 ± 3.77%. SEM analysis showed membranes with uniform and homogeneous surface, with a mean thickness of 68.71 μm. Analysis by AFM revealed roughness around 285 nm. The weight of the membranes ranged from 0.03 to 0.04 g, indicating their uniformity, and the surface pH exhibited a mean of 6.9 ± 0.25, a value close to the pH of the saliva. The Alamar Blue assay showed significant differences in mitochondrial activity between groups at 24 h (L1> C, p = 0.0118) and at 48 h (L1> C, p = 0.0022; L4> C, p = 0.0002; L4>L1, p = 0.0022). The Live/Dead assay showed higher density of live cells in irradiated groups (L1 and L4) compared to the group without irradiation (C), which was confirmed by assay of Annexin V/PI, which showed a greater percentage of viable cells in L4 (89.5%) and L1 (87.0%) compared to C (78.4%) at 72 h. The Ki67 immunoexpression was higher in L4 and L1 and these two groups also showed a higher percentage of cells in the proliferative phases of the cell cycle (S and G2/M). The SEM analysis showed in group C cells with more rounded morphology and with few projections, as well as cell debris, whereas in the irradiated groups the cells exhibited a more flat arrangement, more distributed projections and focal adhesion points, especially in L4. Taken together, the results of the present study shown that laser therapy in the studied patterns, especially at the dose of 4 J/cm², has a positive effect on viability and proliferation of PDLSCs on chitosan membranes, thereby allowing the cells to overcome any adverse effects of the scaffold microenvironment.