The Effect Of Er:YAG Laser Irradiation On Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Cells

Background Periodontal ligament contains a group of complex periodontal ligament cells. These cells have strongheterogeneous,including fibroblasts, osteoblasts, osteoclast as well as undifferentiated mesenchymal stem cells. Periodontal ligament cells are the most common cells in Periodontal ligament, which have a strong self-renew, proliferation and differentiation function. In addition, it also can induce new fibers and cementum formation continuously, plays an important role in bone remodeling and periodontal regeneration. Er.YAG laser is a kind of near infrared laser. It can be absorbed easily by water, generating lesser heat when it is used to irradiate tissue, so results in less damage to the tissue because of its wavelength of 2940 nm. At the same time, Er:YAG laser has a good effect in cutting hard tissues as well as soft tissues. Based on that, Er:YAG laser was supposed to be the most promising laser in periodontal treatment in recent years. Despite the current reports about Er:YAG laser are very common, the effect of Er:YAG laser on human periodontal ligament cells are extremely rare. Many studies suggested that periodontal ligament cells play important roles in periodontal tissue repair. This experiment aimed to observe the proliferation and differentiation of human periodontal ligament cells after the irradiation of different output power, the same irradiation time and the different irradiation time, the same power output Er: YAG laser through MTT assay, colony formation assay, scratch test, Elisa test, Western blot.Objective 1.To compare the effect of different output power and irradiation time of 2940 nm Er:YAG laser irradiation on cells proliferation of human periodontal ligament cells.2.To compare the effect of different output power and irradiation time of 2940 nm Er:YAG laser irradiation on cells differentiation of human periodontal ligament cells.Methods Firstly, the impacted third molars were collected in the Affiliated Stomatological Hospital of nanjing medical university (non-carious, periapical periodontitis and periodontitis with the selected teeth). The periodontal ligament tissues attached to the middle third of the root surface of the canine incisor teeth were gently curetted, removed, cut into small pieces, and plated onto 6-well plate with cover slip in place of the dry anchoring traditionally. When the cells surrounding the tissue explants were confluent, they were subcultured with 0.25% trypsin and collagenase digestion, and transferred to a tissue culture flask. MTT colorimetric assay,Von Kossa staining were used to determine their biological characteristics. Immunocytochemical staining cytokeratin and vimentin were used to identify the source of human periodontal ligament cells. Cells at 3rd to 6th passages were transferred to a tissue culture flask for subsequent experiments. Secondly, the cells were irradiated by Er:YAG laser according to the different output power, the same irradiation time (0 W,0.45 W,0.6 W and 0.75 W, t=10 s), and the different irradiation time, the same power output (0 s,10 s,30 s,60 s, W=0.6 W). The methyltetrazolium (MTT) assay, the clony formation assay, the wound scratch assay, elisa, western blot were performed to observe the effects of Er:YAG laser on human periodontal ligament cells proliferation, clone formation, migration, secretion of TGF-β1 and protein expression which are associated with ossification(such as ALP, OCN, Runx2).Results 1.Under a microscope, we could observe spindle-shaped cells with equal plasm. Immunocytochemical staining show positive for vimentin, negative for cytokeratin. This prove that those cells derived from mesoderm.2. In different power groups,0.45 W and 0.6 W can promote cell proliferation and clony formation, migration rate was faster than the control group, secretion of TGF-β1 was more than the control group (P<0.05), but there is no significant statistical differences between the 0.45 W and 0.6 W. In different irradiation time groups, cell proliferation and clony formation is higher, migration rate was faster than the control group when the irradiation time is 10 s, secretion of TGF-β1 was more than the control group(P<0.05). On the contrary, we get the opposite result when the irradiation time is 60 s(P<0.05). 3. In different power groups,0.45 W and 0.6 W, the expression of ALP, OCN, Runx2 is higher than the control group (laser group) (P<0.05), the expression of ALP and Runx2 up to the maximum at the time of 5 days, the expression of OCN reaches the maximum at the time of 7 days, but there is no significant statistical differences between the 0.45 W and 0.6 W. In different irradiation time groups, the expression of ALP, OCN and Runx2 is higher compared with the control group when the exposure time is 10 s(P<0.05). On the contrary, we get the opposite result when the irradiation time is 60 s.Conclusion l.We could isolate human periodontal ligament cells with enzyme and collagenase digestion in vitro. Combined with microscopic morphology and the source of the tissue, we could confirm that these cells are human periodontal ligament cells.2.A ppropriate amount of Er:YAG laser irradiation could promote human periodontal ligament cells proliferation, clony formation, migration and TGF-β1 secretion.3.Appropriate amount of Er:YAG laser irradiation could promote ALP, OCN, Runx2 expression of human periodontal ligament cells.