Figure 1.

In vitro experimental procedures and real time RT-PCR analysis showing expression of select ECM and inflammatory cytokine genes in PDL-treated VFFs. A: Representative immunostain of VFFs positive for prolyl 4-hydroxylase β (green). Cell nuclei are stained with Hoechst 33342 (blue). B: Results of pilot experimentation using Zap-It laser alignment paper to evaluate variation in spot diameter, beam shape and energy distribution as a function of laser energy and laser-target distance. Uniform energy distribution within a 6.51 mm spot diameter was obtained with 0.8 J laser energy delivered from a 22 mm distance, and these parameters were selected for subsequent experimentation. C: Schematic illustrating PDL laser delivery for the in vitro experiment using the parameters indentified in B. The culture well diameter (6.5 mm) closely matched the laser spot diameter (6.51 mm). D: Photograph illustrating laser energy delivery across each culture well surface with no cross-irradiation of adjacent wells. This image was captured using the 505 nm aiming laser on the PDL instrument. E: Fold change in relative mRNA expression of COL1A1, COL3A1, MMP13, IL1B, IL6, TGFB1 and COX2 in PDL-treated fibroblasts compared to control, 3, 24 and 72 h following delivery of either 4.82 or 9.64 J/cm² laser fluence. Data represent four independent experimental replicates. ACTB was employed as a housekeeping gene.