Efeito da laserterapia em células pré-osteoblásticas MC3T3-E1 cultivadas sobre arcabouços de quitosana

Abstract

Chitosan is a biopolymer that presents potential use as a scaffold in tissue engineering due to its mechanical and biological properties, such as biodegradability, non-toxicity, antibacterial effect and biocompatibility. Laser therapy has been studied as an auxiliary tool in tissue engineering techniques due to its in vitro biostimulating effect on several cell types, but its effect on cells grown on chitosan scaffolds is still unknown. The aim of this study was to evaluate the effect of laser therapy on pre-osteoblastic cells cultured on chitosan scaffolds. Chitosan and membranes produced were subjected to physical-chemical and surface characterization. Cells of the MC3T3-E1 lineage were expanded and divided into four groups according to the surface and the use or not of irradiation: P – plastic of the culture dish, without irradiation, as a positive control of cell growth; L0 – chitosan scaffolds, without irradiation; L1 – chitosan scaffolds, cells submitted to irradiation with a dose of 1 J/cm²; and L4 – chitosan scaffolds, cells submitted to irradiation with a dose of 4 J/cm². Laser therapy was performed with InGaAlP diode laser, with wavelength of 660 nm, power of 30 mW, in a single dose. Cell proliferation was analyzed at 24, 48 and 72 h after irradiation through the Alamar Blue metabolic reduction assay, while cell viability was assessed by Live/Dead and Annexin V/PI assays. Cell morphology and cell-biomaterial integration were evaluated at 72 h by SEM. Data from the proliferation assay showed that L4 group exhibited greater proliferation compared to P and L0 groups at 24 h (p <0.01) and showed a proliferative tendency in relation to L0 and L1 at 72 h. The evaluation of viability by Live/Dead assay revealed a large number of viable cells in all groups, which was confirmed by Annexin V/PI analysis, which showed a high percentage of cell viability in the studied groups (P: 89.2%; L0: 82.1%; L1: 82%; e L4: 85.2%). SEM analysis revealed that in the non-irradiated group (L0) the cells were more isolated and rounded, while in the irradiated groups (L1 and L4) the cells showed more evident projections and formed cellular clusters. Taken together, data from the present study showed that the chitosan scaffolds produced did not influence the proliferation of MC3T3-E1 cells, but also did not impair cell viability. Photobiomodulation, especially at the dose of 4 J/cm², promoted an increase in cell proliferation and allowed the cells to overcome any unfavorable conditions on the surface of the biomaterial. These results suggest that laser therapy may be an effective tool to promote cell proliferation in tissue engineering techniques with chitosan scaffolds.