Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts.

We investigated the biological effects of Er:YAG laser (2940-nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm-2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm-2 promoted maximal cell proliferation, determined by WST-8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post-irradiation. Elevation of ATP level, Ki67 staining, and cyclin-A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post-irradiation at 6.3 J cm-2 , and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up-regulation of 21 genes involved in heat-related biological responses and ER-associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real-time PCR. Surface temperature measurement confirmed that 6.3 J cm-2 generated heat (40.9°C post-irradiation). Treatment with 40°C-warmed medium increased proliferation. Laser-induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm-2 strongly potentiated HGF proliferation via photo-thermal stress, suggesting potential wound-healing benefit.

Increased cell proliferation and differential protein expression induced by low-level Er:YAG laser irradiation in human gingival fibroblasts: proteomic analysis.

Erbium-doped yttrium aluminum garnet (Er:YAG) laser treatment has demonstrated favorable wound healing effect after periodontal therapy. One of the reasons may be the positive biological effect of the low-level laser on the irradiated tissues, although the mechanism remains unclear. The aim of this study was to investigate the effect of low-level Er:YAG laser irradiation on cell proliferation and laser-induced differential expression of proteins in human gingival fibroblasts (HGFs) by proteomic analysis. In the first experiment, HGFs were exposed to low-level Er:YAG laser irradiation and the laser-induced cell proliferation and damage were evaluated on day 3. In the second experiment, proteomic analysis was performed on day 1 after irradiation. The peptides prepared from HGFs were analyzed by a hybrid ion trap-Fourier transform mass spectrometer, Mascot search engine, and UniProtKB database. A significant increase in cell proliferation without cell damage after irradiation was observed. Among the total identified 377 proteins, 59 proteins, including galectin-7, which was associated with the process of wound healing, were upregulated and 15 proteins were downregulated in laser-treated HGFs. In the third experiment, the increase in messenger RNA (mRNA) and protein expression of galectin-7 in the irradiated HGFs was validated by various analytical techniques. In addition, the effect of recombinant human galectin-7 on the modulation of HGFs proliferation was confirmed. The results indicate that low-level Er:YAG laser irradiation can promote HGF proliferation and induce a significant change in protein expression and the upregulation of galectin-7 expression may partly contribute to the increase in cell proliferation.

Gingival tissue healing following Er:YAG laser ablation compared to electrosurgery in rats.

The erbium-doped yttrium aluminum garnet (Er:YAG) laser is currently used for periodontal soft tissue management with favorable outcomes. However, the process of wound healing after Er:YAG laser (ErL) treatment has not been fully elucidated yet. The aim of this study was to investigate the gingival tissue healing after ErL ablation in comparison with that after electrosurgery (ElS). Gingival defects were created in 28 rats by ablation with ErL irradiation or ElS. The chronological changes in wound healing were evaluated using histological, histometrical, and immunohistochemical analyses. The ErL-ablated gingival tissue revealed much less thermal damage, compared to the ElS. In the ElS sites, the postoperative tissue destruction continued due to thermal damage, while in the ErL sites, tissue degradation was limited and the defects were re-epithelialized early. Heat shock protein (Hsp) 72/73 expression was detected abundantly remote from the wound in the ElS, whereas it was slightly observed in close proximity to the wound in the ErL sites. Hsp47 expression was observed in the entire connective tissue early in the wound healing and was found limited in the wound area later. This phenomenon proceeded faster in the ErL sites than in the ElS sites. Expression of proliferating cell nuclear antigen (PCNA) persisted in the epithelial tissue for a longer period in the ElS than that in the ErL. The ErL results in faster and more favorable gingival wound healing compared to the ElS, suggesting that the ErL is a safe and suitable tool for periodontal soft tissue management.

Application of quantitative proteomic analysis using tandem mass tags for discovery and identification of novel biomarkers in periodontal disease.

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)-9 and neutrophil gelatinase-associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.

Effect of human beta-defensin-3 on the proliferation of fibroblasts on periodontally involved root surfaces.

Human beta-defensin-3 (HBD-3) has versatile antibacterial activity against oral bacteria and can promote the proliferation of fibroblasts. The goal of the present study was to investigate the effect of HBD-3 on attachment and proliferation of periodontal ligament cells (PDL) onto the periodontitis affected root surfaces. PDL cells were seeded onto healthy and diseased root specimens with scaling and root planing (SRP), SRP & HBD-3 (100 ng/ml), or SRP & HBD-3 (200 ng/ml) treatment for 1, 3, and 7 days incubation. The results showed that HBD-3, especially in the 200 ng/ml group, significantly promoted fibroblast attachment and proliferation onto the diseased root surfaces. The cell number in the HBD-3 group was much greater than in the group treated with SRP alone. On day 7, the cells in the HBD-3 were well-spread and formed a network similar to those on the surfaces of the healthy root specimens. These results suggest that HBD-3 could play an important role in antibacterial activity and fibroblast proliferation, thus promoting periodontal regeneration. Meanwhile, HBD-3 might act as a potent regeneration-promoter in infectious diseases.