Effect Of Low Level Laser Therapy On Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament

Objective:Periodontitis is a chronic infectious disease leads to destruction of periodontal connective tissue and the bone tissue supporting teeth,and is one of the leading causes of tooth loss in adults.The traditional treatment of periodontitis is mainly to remove local irritants and control inflammation.Low-power laser is a non-invasive treatment method,which has the functions of anti-inflammatory,analgesic and promoting local tissue regeneration,etc.It has been used in the treatment of periodontal diseases,but the effect of LLLT on host cells and specific mechanism need to be further studied.The purpose of this study was to investigate the effects of low level laser irradiation on the proliferation and osteogenic differentiation of periodontal membrane stem cells,and to explore the mechanism of these effects.Methods:1.Periodontal ligament stem cells were cultured by enzymolysis,purified by limited dilution,and identified by flow cytometry,immunofluorescence and multi-directional differentiation induction;2.Periodontal ligament stem cells were irradiated by low-power laser,and their proliferation was detected by CCK-8 method;The expression of PCNA and KI67 was detected by qRT KI-PCR;EdU staining was used to label the proliferating cells;Cell cycle was detected by flow cytometry;The best energy density of laser irradiation was selected;3.Alkaline phosphatase activity and alizarin red staining were used to detect the osteogenic differentiation ability;The expression of BSP,ALP,OCN and COL-1 in PDLSCs were detected by Western blot;4.Transcriptome sequencing was used to find the differentially expressed genes after low power laser irradiation.Go enrichment analysis and KEGG pathway analysis were used to verify the sequencing results.Results:1.Using the method of enzymatic tissue culture,the cells could be seen crawling out from around the tissue at 5-7 days,and the clone community was selected by limited dilution method for further culture;Flow cytometry showed that CD29,CD44,CD90 and CD 105 were positive,while CD34 and CD45 were negative;The positive expression of nesting and STRO-1 was detected by immunofluorescence assay.After osteogenic induction,adipogenic induction and chondrogenic induction,mineralized nodules,lipid droplets and chondrospheres were formed,which indicated that the cultured cells had the ability of multi-directional differentiation.2.CCK-8 method was used to detect cell proliferation and draw cell growth curve;EdU labeled proliferated cells,the results showed that:after 36-60 hours of low-power laser irradiation,the proliferation of periodontal ligament stem cells was promoted in 1 J/cm2s group,but inhibited in 2J/cm2 group;3.qRT-PCR results showed that the expression of proliferation related genes PCNA and KI67 were down regulated in 1 J/cm2 group at 72h after low power laser irradiation,but up-regulated in 2J/cm2 group;4.Under the condition of nm,the energy density of 2J/cm2 group up-regulated the expression of osteogenic related mRNA and protein of PDLSCs;However,under OIM condition,the energy density of 1J/cm2 group down regulated the expression of osteogenic related mRNA and protein of PDLSCs.Conclusion:1.The PDLSCs cultured in the experiment have the ability of self-renewal and cloning,conform to the surface markers of mesenchymal stem cells,and have the ability of multi-directional differentiation of osteogenesis,adipogenesis and chondrogenesis.The cells were proved to be periodontal ligament stem cells.2.The cells in 1 J/cm2 group could promote cell proliferation 36-60 h after irradiation;The proliferation of PDLSCs in 2J/cm2 group was inhibited at 36-60h after irradiation,and promoted at 72h after LLLT irradiation.3.Under the condition of NM,the energy density of 2 J/cm2 group can promote the osteogenesis of PDLSCs;Under OIM condition,the energy density of 1 J/cm2 group could not inhibit the osteogenesis of PDLSCs.4.After LLLT irradiation,DEGs were concentrated in the TGF-β signal pathway and it may be related to the regulation proliferation and osteogenic differentiation of PDLSCs.