The Study Of The Effect Of Low Level Laser Therapy On Titanium Implant Osseointegration

Background and objectiveLaser is defined as light amplification by stimulated emission of radiation.Its english name is laser,which is taken from the initials of each word in its definition.Human have been studying laser for more than 100 years.Since 1990s,the application of laser in the field of stomatology has developed rapidly.Laser can be used not only in the incision of soft and hard tissues in oral and maxillofacial region,the treatment of vascular malformation,tooth preparation,root canal disinfection,removal of breaking instruments in root canal,etc..,but also can be used to alleviate pain,alleviae postoperative reaction and Promote wound repair and regeneration.With the development of oral implantology,laser therapy has been gradually developed in the field of dental implantation,such as preparing dental implant socket with laser,second stage dental implant surgery with laser,and treatment ofperiimplantitis,solving the problems that traditional methods cannot solve.Medically,according to the biological effects of irradiated biological tissues,laser are classified into low level lasers and high level lasers.Different intensity lasers have different applications in the medical field.The study of low level laser therapy(LLLT)has been ongoing for more than 50 years.In 1968,Mester E first reported the nonthermal effects of LLLT on mouse hair growth[1]In recent years,animal experiments haveshown that LLLT can promote the repair and regeneration of bone tissue defect.The process of implant osseointegration is similar to regeneration and repair of bone defect.This involves a variety of biological factors such as BMP.FGF and VEGF,and a series of cells such as osteoblasts and osteoclasts.Among them,osteoblasts play an important role.MC3T3-E1 cells are pre-osteoblasts with osteogenic differentiation ability,which are commonly used in bone repair and regeneration research.Different lasers have different biological effect on the same biological tissue.And the same laser also has different effectson biological tissues due to different laser parameters such as wavelength,power,energy,laser mode,and illumination mode.LLLT mainly uses near-infrared light.Currently,the near-infrared laser commonly used in dentistry includes 810nm semiconductor laser and Nd:YAG laser.Therefore,this study exploredthe effects of low level Nd:YAG laser and low level 810nm semiconductor laser irradiation on proliferation and osteogenesis of MC3T3-E1 cells,and the impacton implant osseointegration,in order to provide reference for the clinical use of LLLT.Materials and MethodsPart I:the Effect of Low Leve1810nm Semiconductor Laser and Low Level Nd:YAG Laser Irradiation on Proliferation and Osteogenic Ability of MC3T3-E1 cells1.Cell culture:The MC3T3-E1 cells frozen in liquid nitrogen were resuscitatd,cultured in culture flask for 2 passages,and plated for experiment when the cells grew and fused to about 80%.2.Cells were divided into 6 groups and labeled as A,B.C,D.E and F.Cells in group A were not irradiated by laser.Cells in group B?D were irradiated by 810nm semiconductor lasers of 200mw,300mw and 500mw respectively.Cells in group E and F were irradiated by Nd:YAG laser of 500mw and 1000mw respectively.CCK-8 assay kit was used to detect the effect of low level laser on the proliferation of MC3T3-E1 cells.ALP activity staining kit was used to detect the effects of laser irradiation on the activity of ALP protein in MC3T3-E1 cells,and qRT-PCR was usedto detect the effects of low level laser on the expression of ALP mRNA,OPN mRNA and RUNX2 mRNA genesin MC3T3-E1 cells.3.After 28 days of laser irradiation,alizarin red staining was used to detect the formation of mineralized nodules in the culture plate.The formation of mineralized nodules in each group was compared by 10%CPC dissolution semiquantitative method.Part II:the Effect of Low LeveI810nm Semiconductor Laser and Low LevelNd:YAG Laser Irradiation on osseointegration of implants in tibia in BeagleDogsFour beagle dogs aged 2 years old were randomly divided into experimental group(2 dogs)and control group(2 dogs).Three Weigao implants were implanted into the bilateral tibias of beagle dogs in experimental group and in one side of the tibia of beagle dogs in control group within 3-9 cm of the talus joint.Immediately after operation,on the third day and the seventh day,the left and right tibia of the experimental group were irradiated by low level Nd:YAG laser or low level 810 nm semiconductor laser respectively,while the control group was not irradiated by LLLT.At the 4th and 8th weeks after operation,one animal in the experimental group and one animal in the control group were executed to make hard tissue sections.After Ladewig staining,sections were observed and photographed under the microscope,and the implant-bone bonding rate(BIC,%)was calculated.Experimental resultsPart ?:the Effect of Low Level 810nm Semiconductor Laser and Low LevelNd:YAG Laser Irradiation on Proliferation and Osteogenic Ability of MC3T3-E1cells1.The morphology of MC3T3-E1 cells was irregular and connected with adjacent cells through several dendritic processes.The expression of ALP protein in cells was detected by ALP protein detection kit when cultured in mineralization induction medium for 7 days.There are brick red mineralized nodules in culture plates after alizarin red staining at 28 days.2.24 hours after laser irradiation,compared with the control group,the proliferation activity of cells irradiated by low level 810nm semiconductor laser and500mw Nd:YAG laser increased significantly(P<0.05).After 48 hours and 72 hours,the contrast gradually weakened.On the 1st,3rd and 7th day after laser irradiation,the expression of RUNX2 mRNA in cells irradiated by low level 810nm semiconductor laser and 500mw Nd:YAG laser gradually increased,and was significantly higher than that in control group.There was no significant difference in the expression of ALP protein in the cells of each group at 7 days after laser irradiation.On the 14th day,the expression of ALP protein in cells which were irradiated by low level 810nm semiconductor laser and 500mw Nd:YAG laser was significantly higher than that in control group(P<0.05)and higher than that in each group at 7 days after laser irradiation.On the 7th and 14th day after laser irradiation,qRT-PCR assay showed that the expression of ALP mRNA in each group was consistent with that of ALP protein activity.After 14 days of laser irradiation,there was no significant difference in the expression of OPN mRNA in each group.On the 14th day,the expression of OPN mRNA in cells irradiated by low level 810nm semiconductor laser and 500mw Nd:YAG laser was significantly higher than that in control group.3.Compared with the control group,28 days after laser irradiation,the formation of mineralized nodules in cells irradiated by low level 810nm semiconductor laser and 500mw Nd:YAG laser was significantly higher than that in the control group.There was no significant difference between the experimental results of the Nd:YAG laser group with 1000mw power and the control group invitro.4.In vitro,proliferation activity,RUNX2 mRNA,ALP protein expression,ALP mRNA,OPN mR-NA and mineralized nodule formation of 500mw 810nm semiconductor laser group and 500mw Nd:YAG laser group were significantly higher than other groups.Therefore,in the second part of the experiment,experiments were carried out using 500mw 810nm semiconductor laser and 500mw Nd:YAG laser.Part II:the Effect of Low Level 810nm Semiconductor Laser and Low LevelNd:YAG Laser Irradiation on osseointegration of implants in tibia of BeagleDogsAt 4 weeks after operation,different degrees of new bone mineralizationappeared in each group of implants.The amount of new bone mineralizationin the low level laser irradiation group was higher than that in the control group.The bone occlusion rate of the 500mw Nd:YAG laser group is 63±12.63%,and 500mw 810nm semiconductor laser group is 64±1.92%,which both are higher than that of the control group(P<0.05).At 8 weeks postoperatively,the osseointegration rates of the two laser irradiation groups were also higher than that of the control group(P<0.05).The BIC of the 500mw Nd:YAG laser group is 72.75±1.83%,and the 500mw 810nm semiconductor laser group is 71.86±2.03%.There was no significant difference between the two laser irradiation groups(P>0.05).Conclusion and significanceThe 810nm low level semiconductor laser at 200mw,300mw and 500mw,and the Nd:YAG laser at 500mw can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells.The 500mw 810nm semiconductor laser andthe 500mw Nd:YAG laser can promote the osseointegration of implants in tibias of beagle dogs,which provides a theoretical basis for the clinical popularization of low level laser in dental implant field.