EFFECT OF LASER THERAPY ON THE BIOLOGICAL ACTIVITY OF PERIODONTAL LIGAMENT STEM CELLS CULTURED ON CHITOSAN SCAFFOLDS
Stem cells; periodontal ligament; biomaterials; chitosan; low-lavel laser irradiation; cell proliferation; cell differentiation.
Oral rehabilitation of patients with extensive bone loss has become increasingly frequent and the gain of bone tissue represents a challenge in the recovery of function. Low-level laser irradiation (LLLI) is capable of stimulating the proliferation of different cell types, including stem cells, but little is known about its efficacy in the proliferation of human periodontal ligament stem cells (PDLSCs) on the surface of polymeric biomaterials. The aim of this study will be to evaluate the influence of LLLI on the proliferation and osteogenic differentiation of PDLSCs cultured on chitosan scaffolds. Cells previously isolated and characterized will be cultured for 24 hours on the chitosan membranes, to ensure cell adhesion, and then divided into three groups: (C) non-irradiated control; (L1) irradiated with a dose of 1 J/cm²; and (L4) irradiated with a dose of 4 J/cm². The irradiations will be performed with an InGaAlP diode laser, with wavelength of 660 nm, power of 30 Mw, tip diameter of 0.01cm² and continuous action mode, in a single dose. Cell viability and proliferation will be evaluated at 24, 48 and 72 hours after irradiation through the Alamar Blue and Live/Dead assays. Cell morphology will be evaluated by MEV at 3, 14 and 21 days and Alizarin Red staining will be used to identify and quantify the formation of calcium deposits on the scaffolds. Biochemical assays will be performed for quantification of alkaline phosphatase levels and the immunolocalization of osteopontin, osteocalcin and bone sialoprotein will be analyzed by indirect immunofluorescence. The quantitative data will be submitted to statistical tests, with significance level of 5%.