| Backgrounds and ObjectivesHyperlipidemia is a systemic disease with higher total cholesterol,higher triglycerides,and/or lower high-density lipoprotein cholesterol.With the improvement of economic level and living standards,the incidence of hyperlipidemia increases gradually.Studies have found that hyperlipidemia is closely related to diseases or processes related to bone regeneration,such as,periodontitis,osteoporosis,implant osseointegration.Ox-LDL(oxidized low density lipoprotein)is one of the biological lipoprotein,which is a key factor to atherosclerosis and osteoporosis.Studies have confirmed that ox-LDL can affect bone regeneration through oxidative stress,inflammatory response,and immune regulation.Periodontal ligament stem cell is one of undifferentiated mesenchymal stem cells in the periodontal ligament.Periodontal ligament stem cells have self-renewal and multi-directional differentiation potential.They can maintain the homeostasis of periodontal tissues and participate in periodontal tissue regeneration.because of the rich source,superior proliferation ability and excellent osteogenic ability,periodontal ligament stem cells have become one of the important seed cells in oral and maxillofacial tissue regeneration progress.Preosteoblasts can differentiate into osteoblasts,which are the main functional cells of bone regeneration and play an important role in the process of bone regeneration.The microenvironment and the activity of the cells are important factors influencing bone regeneration.However,the effects and mechanisms of ox-LDL on osteogenesis in MC3T3-E1 cells and hPDLSCs is still unclear.This project tried to explore the effects of ox-LDL on osteogenesis in MC3T3-E1 cells and hPDLSCs,aiming to reveal the potential effects of the oxidative lipids on bone regeneration.The results might provide a theoretical support for the treatment of bone defect diseases and bone tissue regeneration in patients with hyperlipidemia.Materials and methods1.MC3T3-E1 cells were cultured,cell passage,cell cryopreservation and cellrecovery were also operated.MC3T3-E1 cells were cultured with 0μgg/ml,5μg/ml,15μg/ml，30μg/ml,50μg/ml,100μg/ml ox-LDL for Id,2d,3d,4d,5d and detected with CCK-8 assay kit to reveal the cell proliferation viability.MC3T3-E1 cells were cultured with the osteogenic medium containing 0μg/ml,5μg/ml,15μg/ml,30μg/ml,50μg/ml,100μgg/ml ox-LDL for 3,7,14 days.The expression of Collal,Alpl,Runx2 mRNA were evaluated by qRT-PCR.MC3T3-E1 cells were cultured with the osteogenic medium containing 0μg/ml,50μg/ml ox-LDL for 7,14 days and the activity of alkaline phosphatase were evaluated by micromethod.2.Primary hPDLSCs were isolated and cultured.The immunophenotype of hPDLSCs were analyzed by flow cytometry.Besides,we used osteogenic and adipogenic induction to test the multiple differentiation ability of hPDLSCs.hPDLSCs were cultured and cell passage,cell cryopreservation,cell recovery were also operated.3.hPDLSCs were cultured with Oμg/ml,5μg/ml,15μg/ml,30μg/ml,50μg/ml,100μg/ml ox-LDL for 1d,2d,3d,4d,5d and detected with CCK-8 assay kit to reveal the cell proliferation viability.hPDLSCs were cultured with the osteogenic medium containing 0μg/ml,5μ/ml,15μg/ml,30μg/ml,50μg/ml,100p,g/ml ox-LDL for 3,7,14 days.The expression of COL1A1,ALPL,RUNX2 mRNA were evaluated by qRT-PCR.hPDLSCs were cultured with the osteogenic medium containing Oμg/ml,50μg/ml ox-LDL for 7,14 days and the activity of alkaline phosphatase were evaluated by micromethod.Results1.MC3T3-E1 cells were successfully cultured and cell passage,cell cryopreservation,cell recovery were also completed.The results of CCK-8 assay kit showed no differences between the blank control group and the ox-LDL groups.The expression of Collal,Alpl,Runx2 mRNA decreased in 50μg/ml and 100μg/ml ox-LDL group after 3 days osteogenic induction of MC3T3-E1 cells.The expression of Collal,Alpl,Runx2 mRNA decreased in 15μg/ml,30μg/ml,50μg/ml,100μg/ml ox-LDL groups after 7 days osteogenic induction of MC3T3-E1 cells.When the induction lasted to 14 days,the expression of Collal,Alpl,Runx2 mRNA decreased only in 50μg/ml and 100μg/ml ox-LDL group with significant difference.The activity of alkaline phosphatase decreased in 50μg/ml ox-LDL group after 14 days osteogenic induction of MC3T3-E1 cells.2.hPDLSCs were successfully isolated from pericementum.hPDLSCs expressed high mesenchymal stem cells specific surface markers and contained osteogenic and adipogenic differentiation ability.hPDLSCs were successfully cultured and cell passage,cell cryopreservation,cell recovery were also completed.3.Compared with the blank control group,the CCK-8 value increased slightly in the ox-LDL groups in the third day and this trend continued to the fifth day.The expression of COL1A1 mRNA decreased in all of the ox-LDL group after 3,7,14 days osteogenic induction of hPDLSCs and the decrease in 14d was the most significant.The expression of ALPL、RUNX2 mRNA decreased in 50 μ g/ml、100 μ g/ml ox-LDL group after 3,7 days osteogenic induction and decreased in all of the ox-LDL groups after 14 days osteogenic induction in hPDLSCs.The activity of alkaline phosphatase decreased in 50pμg/ml ox-LDL group after 7 and 14 days osteogenic induction of hPDLSCs.ConclusionsOx-LDL under the concetration of 100 μ g/ml has no effects on the proliferation of MC3T3-E1 cells.Ox-LDL over the concetration of 50 μg/ml inhibit the expression of CollaI,Alpl,Runx2 mRNA and the activity of alkaline phosphatase,inhibiting osteogenic differentiation in MC3T3-E1 cells.Ox-LDL over the concetration of 5 u g/ml slightly promote the proliferation of hPDLSCs.Ox-LDL over the concetration of 50 μg/ml inhibit the expression of COL1A1,ALPL,RUNX2 mRNA and the activity of alkaline phosphatase,inhibiting osteogenic differentiation in hPDLSCs.The above conclusions confirm the negative effect of oxidatively modified lipids on bone regeneration at the cellular level and provide a theoretical support for the treatment of bone defect diseases and bone tissue regeneration in patients with hyperlipidemia. |
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