Brasenia-inspired Hydrogel With Sustained And Sequential Release Of BMP And WNT Activators For The Bone Regeneration

Bone defect caused by fracture,tumor and osteoporosis is a common clinical problem,which seriously affects patient quality of life.Among various methods of repairing bone defects,the gold standard is autologous bone transplantation.However,there are still some problems in autogenous bone transplantation,such as limited bone mass and secondary trauma,which limit its clinical application.In order to overcome these problems,the strategy of tissue engineering bone regeneration has emerged and became one of the research focuses in the past decades.Bone morphogenetic protein(BMP)is widely used because of its strong osteogenic ability,and is the only commercially available therapeutic agent as a bone graft substitute.However,studies have shown that the clinical application of BMP in the treatment of bone defects may produce side effects,or even lead to treatment failure.In addition to ectopic bone formation and inflammation,it may even lead to osteolysis,which makes bone healing inefficient,and even increases the risk of microfracture.BMP and WNT signal pathways play key roles in bone development,homeostasis and regeneration,but there are complex positive and negative interactions between them.Therefore,it is a more effective way to achieve bone regeneration to find appropriate means and methods to activate BMP and WNT signal pathways,make them play a synergistic role,and simultaneously offset the mutual inhibition of the two signal pathways.Due to the high cost and short half-life of proteins,this study intends to use small molecular compounds to activate signal pathways.In Chapter 1,we described the background and basis of this research,reviewed the research progress and problems of seed cells,growth factors and scaffold materials in bone tissue engineering,and introduced the research ideas of this topic:for the problems of bone tissue engineering in the process of bone defect repair,inspired by the special double-layer structure of the stem of Brasenia schreberi,It is planned to use sodium alginate(ALG)and chitosan(CS)to synthesize double-layer hydrogels,and load small molecule drugs that can activate BMP and WNT signal pathways,so that they can release successively and activate corresponding signal pathways sequentially.In Chapter 2,we prepared ALG/CS hydrogels by crosslinking method,and characterized their surface morphology,mechanical properties,degradation and drug release properties in vitro by scanning electron microscopy,compression strength test,degradation test and release test.The results showed that the inner and outer layers of the synthesized ALG/CS hydrogels had different structures,which could support small molecule drugs and achieve the goal of sequential and slow drug release.In Chapter 3,we first screened the application concentration of small molecule drugs,determined the action mode of drugs,and then prepared different groups of drug-loaded ALG/CS hydrogels.The effects of drug-loaded ALG/CS hydrogel on the differentiation of mouse calvarial preosteoblasts were detected by ALP staining,alizarin red staining and q RT PCR.The results showed that the synthesized drug-loaded ALG/CS hydrogels of different groups could promote the osteoblastic differentiation of osteoblasts in vitro to varying degrees.Among them,the drug-loaded ALG/CS hydrogels that released FK506 first and then BIO could promote the osteoblastic differentiation more significantly than other groups.In Chapter 4,we first evaluated the biosafety and biocompatibility of drug-loaded ALG/CS hydrogel through subcutaneous implantation model and local bone defect implantation model;Secondly,two models of rat calvarial critical bone defect and rat femoral critical bone defect were established.Four weeks and eight weeks after implantation of drug-loaded ALG/CS hydrogel,the formation of new bone and the quality and quantity of new bone in the bone defect area were evaluated by micro-CT and histology respectively.The results showed that the local application of the synthesized drug-loaded ALG/CS hydrogel showed good biosafety and biocompatibility.Different groups could promote the repair of cortical and cancellous bone defects.Among them,the drug-loaded ALG/CS hydrogel that released FK506 first and then BIO could accelerate the repair of bone defects and the maturation of bone tissue compared with other groups.In Chapter 5,we preliminarily discussed the possible mechanism of drug-loaded ALG/CS hydrogel promoting osteoblast differentiation in vitro and bone defect repair in vivo.Through Transwell migration experiment and Western blot detection,it was clear that drug-loaded ALG/CS hydrogel could promote the migration of BMSCs to varying degrees,which may be the reason why it recruited stem cells to reach the bone defect area and promote bone regeneration in the implanted bone defect area in vivo;In addition,the drug-loaded ALG/CS hydrogel can release small molecule drugs in the outer and inner layers in order to activate BMP and WNT signal pathways sequentially,which may play a synergistic role in the bone defect repair process and eliminate the mutual inhibition between BMP and WNT signal pathways.In conclusion,this study prepared a drug-loaded ALG/CS hydrogel with a structure similar to that of Brasenia,and successfully achieved the sequential and slow release of BMP signal pathway activator and WNT signal pathway activator.The biosafety and biocompatibility of drug-loaded ALG/CS hydrogel were evaluated through in vitro and in vivo studies,as well as the role and mechanism of promoting osteocyte differentiation and new bone formation in vitro and in vivo,which laid a theoretical and experimental basis for clinical treatment of bone defect destructive diseases and accelerating bone damage repair.