Sequential Release Of BMP/WNT Signaling Activators By Chitosan/Sodium Alginate Hydrogel Promotes Osteogenic Differentiation In Vitro

Bone defects can be caused by accidental trauma,chronic inflammation,and bone tumors,etc..For the bone defect of oral and maxillofacial region,it not only affects the function of chewing and articulation,but also affects the aesthetics,as well as physiological and mental health of the patients.Therefore,finding an effective method to treat bone defects has become a hot topic of research all over the world.As the "gold standard",autologous bone graft and allograft have limited sources,poor shaping and immune rejection,so bone tissue engineering has shown great potential in repairing bone defects and promoting bone regeneration.For cytokines in tissue engineering,BMP and WNT signaling pathways are known to be two important signaling pathways in bone formation and regeneration.However,the roles of BMP or WNT alone in bone defect repair is limited.Studies have shown that in the dynamic process of bone formation and bone regeneration,these two signaling pathways interact with each other in a complex way.During the dynamic process of bone formation,the increased expression of WNT signal is conducive to the renewal of stem cells,while the activation of BMP signal can promote the differentiation of osteoblasts.Therefore,we intended to construct a double-layer composite drug carrier material,which activates BMP and WNT signaling pathways through sequential release,which may achieve the purpose of exerting the synergistic effects of the two signaling pathways to offset the antagonistic effects.Chitosan and sodium alginate are widely used as drug carriers because of their good biocompatibility,promoting cell adhesion and bone matrix mineralization.In addition,due to the high price of BMP protein and other cytokines,they cannot be applied in large quantities.We intended to apply small molecule compounds with the same or similar effects,FK506 and BIO,which are activators of BMP and WNT signaling pathways,respectively,which can promote osteogenic differentiation of cells.Therefore,the purpose of this study was to construct a chitosan/sodium alginate bilayer composite to load BIO and FK506 to achieve the purpose of sequential releaseand sequential activation of the signaling pathway,and to study its roles in promoting osteogenic differentiation of MC3T3-E1 cell lines in vitro,so as to provide new ideas and theoretical support for clinical bone repair and bone regeneration in the future.In chapter 2,in order to better exert the roles of BMP and WNT signaling pathways in promoting osteogenic differentiation of pre-osteoblasts,we determined the optimal concentrations of BMP signaling pathway activator FK506 and WNT signaling pathway activator BIO.We determined the concentration ranges of FK506 and BIO by CCK-8,in which they had no effect on the viability of pre-osteoblast cell line MC3T3-E1 cells.The influences of signal pathway activators on the osteogenic differentiation of MC3T3-E1 cells within the safe concentration ranges were detected by ALP and alizarin red staining.We found that FK506 had a stronger effect on early alkaline phosphatase activity than BIO,with 2 ?g/ml of FK506 exhibiting the strongest effects on promoting ALP activity.Alizarin red staining results showed that0.1 ?M of BIO showed the strongest calcium nodule formation.In chapter 3,to sequential activate BMP and WNT signaling pathways,we prepared a dual layer material with outer sodium alginate and inner chitosan.The surface morphology was observed using scanning electron microscope(SEM).Weighing method was used to determine the degradation curve of the material.The results showed that chitosan/sodium alginate composite scaffold slowly degradated until 35 days.To measure the release of the drugs,we prepared inner chitosan and outer sodium alginate composite with inner BIO and outer FK506,respectively.Ultraviolet spectrophotometric method was used to determine the release of the BIO and FK506.The results showed that FK506 and BIO released sequentially,with outer FK506 release within 14 days,and the inner BIO beginning to release on the 5th day,and finishing releasing 48 hours later.In chapter 4,in order to further validate our hypothesis,we prepared the dual layer materials loaded with different drugs.ALP staining and alizarin red staining was used to detect the osteogenic differentiation of MC3T3-E1 cell line.Real-time RT-PCR was used to detect the expressions of osteogenic genes.We found that the experimental group with internal BIO and external FK506 showed higher alkaline phosphatase activity,more calcium nodule formation,and significantly enhanced the expressions of osteoblastic genes Runx2 and Ocn,compared with other groups.In conclusion,we synthesized a sodium alginate/chitosan double layer compositescaffold with stable structure.The drug loading materials with inner BIO and outer FK506 could release sequentially and promote the osteogenic differentiation of MC3T3-E1 cells in vitro.Our results provided a new idea for bone regeneration of clinical bone defects.