The Polypeptide OP3-4 Induces Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Via AKT/GSK3β/β-catenin Pathway And Promotes Mandibular Defect Bone Regeneration

Background and purposeMandibular defect badly affects patient health as a familiar deformity.Congenital malformations,tumors,tumor surgery and trauma are the main causes of mandibular defects.Although bone grafting is an effective repair method,the traditional repair methods such as autologous bone graft,allogeneic bone graft,and distraction osteogenesis still face the problems of limited number of donors,strong immune response and long treatment period.In recent years,studies have found that peptides have obvious advantages in the process of exerting biological activity.They can be efficiently used by the body in the form of active absorption.At the same time,they can also improve element absorption and mineral transport,promote growth and regeneration,and immune regulation.OP3-4（YCEIEFCYLIR）,as a polypeptide that mimics osteoprotegerin（OPG）,and can inhibit osteoclast production,thereby inhibiting the process of bone resorption and promoting bone regeneration.However,its relevant mechanism is still not fully clarified,and further research is required.As a type of mesenchymal stem cells（MSCs）,bone marrow mesenchymal stem cells（BMSCs）can participate in bone remodeling through osteogenic differentiation.Moreover,studies have also found that MSCs express RANKL.Therefore,this subject intends to explore the mechanism by which the polypeptide OP3-4 promotes the osteogenic differentiation of BMSCs through related experimental design,thereby promoting the regeneration of mandibular defects.The expected results will provide new ideas for the treatment of mandibular defects and the application of OP3-4 in the field of bone regeneration,which has positive scientific significance.Materials and methods1.The first part is an in vivo experiment.By constructing a rat mandibular defect model,the grouping of all 8-week-old male rats were random,which countaining the control group,hydrogel group and hydrogel loaded OP3-4 group.2 and 4 weeks after the establishment of mandibular defect model,the rats were sacrificed by cardiac perfusion under general anesthesia and the mandibular was obtained as the object of histomorphological evaluation.Hematoxylin and eosin（HE）staining and Masson staining was performed on paraffin sections to observe bone regeneration of the mandibular defect;immunohistochemistry（IHC）staining was used to detect alkaline phosphatase（ALP）,runt-related transcription factor（Runx2）and type I collagen（Coll）positive expression,and explore the effect of OP3-4 on mandibular defect bone regeneration.2.The second part is an in vitro experiment.The aim was to explore the mechanism of OP3-4 in promoting osteogenic differentiation of BMSCs by extracting rat BMSCs.BMSCs were given 200μM OP3-4 under osteogenic induction conditions,and the expression of Runx2 and Osterix were evaluated by western blot.The protein kinase B（AKT）inhibitor LY294002 was added to further verify the expression of Runx2 and Osterix and the signal pathway related factors AKT,glycogen synthase kinase 3β（GSK3β）and β-catenin under the conditions of osteogenic induction,and to verify whether OP3-4 promotes the osteogenic differentiation of rat BMSCs through AKT/GSK3β/β-catenin pathway.Results1.Two weeks after the rat mandible defect,HE staining and Masson staining revealed that the hydrogel loaded OP3-4 group showed more regular fiber arrangement and slight increase of scattered punctate or needle bone tissue compared with the control and hydrogel groups.Four weeks after surgery,substantial new bone appeared in the hydrogel loaded OP3-4 group,the hydrogel showed degradation,and the structure became sparser.IHC staining showed a significant increase in the positive expression of ALP and Runx2 in the hydrogel loaded OP3-4 group as compared to the control and hydrogel groups.And the hydrogel loaded OP3-4 group showed positive expression of Coll.2.Western blot results showed that when rat BMSCs were cultured with osteogenic induction medium containing 200μM OP3-4,the expression of Runx2 and Osterix were significantly increased compared with the other three groups.After adding AKT inhibitor LY294002,the expression of Runx2,Osterix,p-AKT/AKT,p-GSK3β/GSK3β,andβ-catenin were significant reduced.ConclusionThe polypeptide OP3-4 induces BMSCs osteogenic differentiation through the AKT/GSK3β/β-catenin pathway and promotes the regeneration of mandibular defects.