| BackgroudThe periodontal ligament(PDL)is the connective tissue between the root and alveolar bone.The teeth are suspended in the alveolar fossa by PDL,which sustains,supports,transfers and distributes the occlusal force from the teeth continuously.Under the occlusal force,the changes of various cells and key molecules in PDL can be triggered and activated,thus affecting PDL shaping and remodeling.Tooth avulsion is common in oral emergency.PDL and dental pulp are injured completely.When patients visit the clinic,the periodontal ligament is often damaged or even necrotic.Pathological healing and even tooth loss are easily formed after replantation treatment.The treatment of Tooth avulsion is difficult and the prognosis is very unstable.How to achieve ideal healing of PDL of replanted teeth is still a problem for the management of dental trauma.Among the factors which affected the healing of replanted teeth,the time of fixing avulsed teeth and the timing of occlusal loading have great influence on PDL healing of the avulsed teeth.Andreasen,the founder of dental trauma research,proposed the concept of functional fixation which based on clinical practice.He emphasized that maintaining the physiological mobility of avulsed replanted teeth is essential for the regeneration and healing of PDL.In fact,periodontal tissue is one of the most active tissues in human reconstruction,which is related to its masticatory function and occlusal pressure.However,up to now,the molecular mechanism of occlusal force acting on PDL and promoting the regeneration and reconstruction of damaged PDL is still unclear.Our previous studies found that dynamic pressure stimulation on the periodontal ligaments stem cells(PDLSCs)could lead to the express of the insulin like growth factor-I isoform,mechano-growth factor(MGF).MGF is an alternative splicing of IGF-1,expressed in many tissues and encodes a unique E-domain region,which has ability of tissue regeneration.However,molecular mechanisms of MGF were not fully understood.Many researches reveal evidence to support the notion that biologic actions and signaling of the MGF was mediated via an IGF-1R–independent mechanism.Accordingly,the receptor proteins and signal transduction pathway of MGF are worthy of exploration.The function and molecular mechanism of MGF regulatied the differentiation of PDLSCs under pressure were evaluated both in vitro and in vivo,whcih provide new strategies and ideas for obtaining a satisfactory periodontal healing for avulsed teeth after replantation.Objectives(1)To clarify the effects of pressure and MGF on the proliferation,migration,adhesion,collagen synthesis and differentiation of PDLSCs and PDL cells;(2)To elucidate the molecular mechanism of pressure and MGF regulating PDLSCs;(3)To verification of the role of MGF in the regeneration and restoration of periodontal ligament under occlusal force in vivo;Methods(1)PDLSCs were separated by immunomagnetic beads.The PDLSCs and periodontal ligament cells were identified by clone formation test,cell surface markers identification,multiple differentiation potential test and growth curve determination.(2)PDLSCs and PDL cells were treated by pressures and MGF with different concentrations.Cell viability were detected by CCK8.Real-time PCR and Western blotting were used to detect the expression of PCNA,CXCR4,CAP,Osterix,BSP,OPN,CEMP1,Scleraxis,COL-Iα1 and COL-IIIα1.(3)The receptor of MGF was tested by antibody array technology,and the signal pathway of pressure and MGF acting on PDLSCs was analyzed by antibody array combined with Western blotting.(4)A delayed replantation model of avulsed teeth in rats was established;MGF was given during replantation and deprive the occlusal force by feeding soft food.Micro-CT scan and HE staining were used to detect root resorption rate and periodontal ligament healing of the replanted teeth.The effects of MGF in periodontal tissue regeneration and repair under occlusal force were verified in vivo by Micro-CT examination and pathological examination.Results(1)PDLSCs sorted by immunomagnetic beads were positive for stem cell surface markers.PDLSCs also have strong ability of cloning and multidirectional differentiation.However,PDL cells were negative for stem cell surface markers,and the ability of cloning and multidirectional differentiation was poor.(2)The expression of Scleraxis in PDLSCs was up-regulated by pressure and MGF,which promoted the PDLSCs differenting into PDL fibroblasts.(3)Pressure and MGF activated Lyn and Fyn kinase which is member of the non-receptor tyrosine Src kinase family and then promoted phosphorylation of 4E-BP1,AKT,ERK1/2 and p38 for signal transduction.(4)Micro-CT scan and HE staining showed that the area of pathological healing of hard food combined with MGF was significantly smaller than that in those by hard food group,soft food group and soft food combined with MGF group.The area of periodontal ligament in hard food combined with MGF group was higher than that in other groups,which the collagen fibers in periodontal ligament were arranged neatly.ConclusionsPressure and MGF can promote PDLSCs differentiation into periodontal ligament fibroblasts,which mainly depends on Lyn and Fyn activation,then regulates downstream phosphorylation of 4E-BP1,AKT,ERK1/2 and p38.Occlusal force and MGF can promote periodontal ligament healing and inhibit pathological healing following delayed replantation of avulsed teeth in rats,... |
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