Analysis of Cell Viability and Gene Expression After Continuous Ultrasound Therapy in L929 Fibroblast Cells

Abstract

Objective This study aimed to analyze cell viability and gene expression interleukin 6 and vascular endothelial growth factor after continuous ultrasound therapy of 1 and 3 MHz in L929 fibroblast cells. Design The L929 cells were cultivated in 12-well plates and divided into the following five groups: Group 1 (G1), nonirradiated; G2, 0.2 W/cm2–1 MHz; G3, 0.5 W/cm2–1 MHz; G4, 0.2 W/cm2–3 MHz; and G5, 0.5 W/cm2–3 MHz. The cells were irradiated at 24 and 48 hrs. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The gene expression analysis was assessed using real-time polymerase chain reaction. Results The G2 and G3 showed a decrease in cell viability when compared with the G1 at 48 hrs (P < 0.01). The G4 and G5 presented an increase in viability (P = 0.01 and P = 0.03, respectively) in 24 to 48 hrs. The cells irradiated at an intensity of 0.5 W/cm2–3 MHz at 48 hrs showed a 0.21-fold decrease in interleukin-6 gene transcripts and a 1.67-fold increase in vascular endothelial growth factor gene transcripts. Conclusions Continuous ultrasound therapy with a frequency of 3 MHz at an intensity of 0.5 W/cm2 stimulates cell proliferation, decreases interleukin-6 gene expression, and increases vascular endothelial growth factor gene expression in L929 fibroblast cells.

Effect of Therapeutic Ultrasound on the Mechanical and Biological Properties of Fibroblasts

“…These studies suggest that every cell responds differently to the ultrasound stimulation dosages and the modality (pulsed or continuous) of application. Then, prolonged continuous ultrasound stimulation at a high dose and frequencies near kilohertz produces a heating effect that may affect cell integrity [31,71], and pulsed treatment at low or high intensities with kilohertz or megahertz of frequency generates a mechanical effect that may positively alter the cell functions while maintaining its viability [25,72]. These reasons justified the dosage prescribed in our study: pulsed mode (50% duty cycle), low (1.0 W/cm 2 ) and high (2.0 W/cm 2 ) intensities, and short time of application of therapeutic stimulation at 1 MHz.…”

“…To this point, we confirmed the hypothesis that therapeutic ultrasound alters the mechanical properties of ligament fibroblasts by cytoskeleton reorganization without harmful effects. Then, the elastic modulus and harmonics are predictive biomarkers of ligament fibroblasts specialized biological functions related to the healing process, such as proliferation, migration, and formation of a new extracellular matrix [23,71,[73][74][75].…”

Cytotoxicity, cytoprotection and morphological analysis of MTA, MTA Repair HP and Biodentine

“…Cytotoxicity was accessed by MTT method (Stockert et al, 2018;Bertin et al, 2019). MTT salt was dissolved in sterile phosphate buffer solution pH 7,4 (0,5 mg mL -1 ).…”

Effect of therapeutic ultrasound on the mechanical and biological properties of fibroblasts

“…For example, when applying therapeutic ultrasound at 1 MHz and low intensities (less than 1.0 W/cm 2 ), cell proliferation and extracellular matrix (ECM) improve on dermal and periodontal ligament fibroblasts [23][24][25][26][27]. Moreover, this high frequency may modulate cell cytoskeleton organization [28,29] which in turn stimulates cell migration [30].…”