Menaquinone 4 Promotes The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells Via The Wnt/β-catenin Signaling Pathway

In the process of orthodontic treatment,after removing bands and brackets from teeth,the moved teeth are often in a precarious state.The reconstruction process of the moved teeth,surrounded by both soft and hard tissues,has not been completed.The moved teeth still tend to revert to their pre-correction state.In the clinical aspects of orthodontics,to consolidate the curative effect and keep teeth at an ideal aesthetic and functional position,a retainer is often worn after removal of a fixed appliance.However,because of the long-wearing cycle of the retainer and patients’ non-cooperation,the treatment effect is often unpredictable.In recent years,exploring the influencing factors of accelerating the process of bone reconstruction of periodontal tissue after orthodontic treatment,inhibiting the restoration of teeth and preventing the relapse after orthodontics,has become a hot topic for orthodontic scholars.The hPDLSCs are a kind of adult stem cells involved in orthodontic tooth movement and periodontal bone remodeling.They are capable of high self-proliferation and renewal,and they have the potential to differentiate into a variety of cells.hPDLSCs can be induced into alveolar bone,cementum and periodontal ligament-like tissue in vitro,which has been regarded as the most promising sources to achieve the regeneration of periodontal tissue.Therefore,research on how to promote the osteogenic differentiation of hPDLSCs and further promote the bone remodeling of periodontal tissue has also become a hot spot in the field of periodontal tissue engineering.Vitamin K2 is a metabolite of intestinal bacteria in the body.It plays a non-negligible role in blood coagulation,bone formation and bone remodeling in mammals,which can directly play a part in the human body and is widely used clinically to treat and prevent osteoporosis.Menaquinone 4(MK-4)is one of the most active form of vitamin K2,whose molecular formula is C31H40O2.Studies have confirmed that in the process of bone metabolism regulation,vitamin K2 can promote bone formation and bone mineralization,inhibit bone resorption through the regulation on gene,molecule and cell levels.Therefore,it can be speculated that MK-4 can enhance the proliferation and osteogenic differentiation potential of hPDLSCs.At present,there has no relevant research reported on the role of MK-4 in the proliferation and osteogenic differentiation of hPDLSCs at home and abroad.Wnt/β-catenin signaling pathway is a classic signaling pathway closely related to bone growth and development,which participate extensively in basic physiological processes such as body development,organ formation and stem cell differentiation.Studies have pointed out that activating the Wnt/β-catenin signaling pathway can promote the osteogenic differentiation of hPDLSCs.What’s more,XAV-939 is a specific inhibitor of the Wnt/β-catenin signaling pathway,which has been applied in many studies.It can provide strong evidence that the Wnt/β-catenin signaling pathway participates in a variety of biological and pathological regulatory responses.Therefore,we speculate that MK-4 can promote the osteogenic differentiation of hPDLSCs mediated by the Wnt/β-catenin signaling pathway,thereby accelerating the bone reconstruction process of peri odontal tissue,inhibiting the restoration of teeth and preventing the relapse after orthodontics.Objectives:The main research direction of the present study was to investigate the role of MK-4 on the proliferation and osteogenic differentiation of hPDLSCs under in vitro culture conditions,and to further explore the role of Wnt/β-catenin signaling pathway in the process of MK-4 enhancing the osteogenic differentiation potential of hPDLSCs after determining its optimal concentration.With the support of the above conclusions,it was hoped that it could be used as a theoretical support to promote the transformation of MK-4 in orthodontic clinicsMaterials and methods:(1)The tissue block method was used to isolate and culture the primary hPDLSCs,and with the support of the limiting dilution cloning method,the cells were further purified.After subculture,3 to 5 generations of hPDLSCs with good growth status were subjected to subsequent experiments.(2)The osteogenic differentiation and adipogenic differentiation induction experiments were performed on hPDLSCs,and the expression of surface markers were detected by flow cytometry.(3)CCK-8 was used to detect the effect of MK-4 on the proliferation of hPDLSCs.ALP activity kit was used to detect the effect of MK-4 on the osteogenic differentiation of hPDLSCs.Based on the above results,the optimal concentration of vitamin K2 was screened out.(4)The effect of the selected MK-4 on the proliferation and osteogenic differentiation of hPDLSCs was re-verified by plate clone formation experiment and Alizarin Red staining(5)The expression of osteogenic differentiation-related genes ALP,Runx2,OCN,and Osterix was detected by qRT-PCR and Western Blot to explore the effect of MK-4 on hPDLSCs.To preliminarily determine the participation of Wnt/β-catenin signaling pathway in this process,the expression level of β-catenin was detected by Western Blot.(6)XAV-939 was used to examine the part of Wnt/β-catenin signaling pathway in the process of MK-4 promoting the osteogenic differentiation of hPDLSCs.Results:(1)The hPDLSCs were successfully isolated and purified,and the 3 to 5 generations after subculturing were in good growth condition.(2)The hPDLSCs were successfully induced to form mineralized nodules and lipid droplets.The markers of CD44,CD90 and CD 105 showed high expression rates,while CD34 and CD45 expression rates were low.(3)MK-4 within a certain concentration range can significantly promote the proliferation of hPDLSCs,and the ALP activity test showed that MK-4 at the concentration of 10-5M can most significantly promote the osteogenic differentiation of hPDLSCs(P<0.05).(4)For the 10-5M MK-4,the results of plate clone formation experiment proved that it can facilitate the proliferation of hPDLSCs,the Alizarin Red staining proved that it can promote the osteogenic differentiation of hPDLSCs.(5)MK-4 at the concentration of 10-5M can observably increase both the mRNA and protein expression levels of ALP,Runx2,OCN and Osterix.It can also observably increase the protein expression level of β-catenin.(6)After application of XAV-939,the conduction of Wnt/β-catenin signaling pathway was inhibited,and the protein expression levels of Osterix,Runx2 and ALP were significantly reduced(P<0.05).So did the p-GSK3β/GSK3β,β-catenin and LEF1(P<0.05).Conclusion:(1)In vitro,MK-4 can observably facilitate the proliferation of hPDLSCs,and can also observably facilitate its osteogenic differentiation.Besides,10-5M MK-4 can exert a satisfactory effect.(2)The Wnt/β-catenin signaling pathway can be activated under the stimulation of MK-4,and its transduction can be also improved,which ultimately improves the osteogenic differentiation ability of hPDLSC.