| BackgroundTissue engineering materials prepared through physical,chemical and biological factors provide a heterogeneous repair strategy for bone repair.The personal designed physical surface morphology has better stimulation persistence than chemical and biological factors.The intensity of the stimulus can be adjusted by the size and shape.It has been widely used in orthopedics,stomatology and other fields.However,most biomaterials cannot be efficiently manipulated and changed after being implanted in vivo,and they lose control of cell proliferation and differentiation.Moreover,bone repair is a long process.Different stages require cells to perform different functions to get the maximum accumulation of bone mass and quality.In addition,different physical morphologies can regulate different behaviors of cells.In order to more efficiently regulate cells,biomaterials need to have controllable physical morphologies to give the cells instructions to maintain stemness or differentiation at a specific time to promote bone repair.Shape memory polymers(SMPs)can sense external stimuli and have shape memory capabilities.The characteristics of biocompatibility and shape memory around body temperature can be achieved by adjusting the composition and feeding ratio.The morphology of the material can be controlled by external light,heat,magnetic and electrical stimulation,so as to achieve the remote control after implanted.Cells make different fate decisions by sensing the specific changes of the extracellular matrix(ECM).For example,a smooth interface promotes cell adhesion and proliferation,while a rough interface promotes differentiation.Therefore,SMPs are used to personalize the morphology of the interface contacted by the cells at different times,and the morphological changes are used to give cells powerful instructions to regulate their rapid differentiation after adhesion and proliferation,which is expected to become the potential solutions of bone defects repair.AimIn this project,shape memory polymers are used to construct a dynamic topological interface,which provides a new solution for the efficient repair of bone defects.It provides an innovative idea for the clinical application of shape memory materials.Methods and ResultsIn this study,polycaprolactone(PCL)was used as the main ingredient to construct a shape memory dynamic topological interface.The research is described in four chapters:the evaluation of the preparation and performance of shape memory materials,the evaluation of biological performance of the dynamic interface,the mechanism of the shape memory dynamic interface regulating cell behavior and the construction and application of a 4D printed bilayer smart periosteum.First,polycaprolactone diacrylate(PCLDA)was prepared by modification with polycaprolactone,and the yield of PCLDA reached 93%.The shape memory polymer prepared by blending PCLDA with a molecular weight of 10,000 and PCLDA with a molecular weight of 2,000 was detected by Differential Scanning Calorimetry(DSC)and its stimulus response temperature was 31-55°C.When the feeding ratio is 5:5,the stimulus response temperature is 46°C.Through dynamic thermomechanical analysis(Dynamic thermomechanical analysis,DMA)analysis,it can be seen that the fixation and recovery effect is 97%,indicating that the polymer synthesized by this method has excellent shape memory ability.The results of cell death staining show that the material has good biocompatibility.By heating in PBS at 46°C,the macroscopic spiral structure can be constructed by twisting,and the micropillar can be fixed from the initial height of 10.0±0.5μm to 2.5±0.5μm by compression,and the height after stimulation is9.8±0.4μm.Besides,the grooves can be fixed to different widths by stretching,and there is no significant difference in the height of the rigid and the width of the groove after recovery.Subsequently,this subject further verified that the dynamic topological interface of shape memory plays a role in regulating cell behavior.Can the rapid accumulation(adhesion and proliferation)of the bone marrow mesenchymal stem cells on the interface and their efficient osteogenic differentiation be achieved? The results of live-dead staining showed that the cells did not appear obvious cell death after short-term stimulation at 46°C.The long-term fluorescence results and CCK-8 test showed that there was no significant difference in cell proliferation at 1,3,and 5 days compared with the unheated group.It shows that the temperature stimulus has no effect on the subsequent dynamic topology test.Compared the fluorescence expression of the proliferation-related protein Ki67 and the Alarmar blue test on the cells in the dynamic group(Dyn),static pillar group(Sta-P)and static flat group(Sta-F).The expression of osteogenic genes Col-1,RUNX2,OCN was detected by RT-PCR.Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenic differentiation effect of the total cells.The results showed that the dynamic interface greatly promoted the osteogenic differentiation of bone marrow mesenchymal stem cells.Through quantitative analysis of HE and Masson III stained sections in vivo,it was found that the dynamic interface had more new bone formation compared with Sta-F and Sta-P groups.Subsequently,we conducted an in-depth analysis of the mechanism of dynamic topological interface regulating cell fate.First,observe the cell shape and internal structure(cytoskeleton distribution and nuclear shape)through Scanning Electronic Microscope(SEM)and Confocal Laser Scanning Microscope(CLSM).The results showed that the cells of the Dyn group were stretched and the nucleus was deformed.Through fluorescent staining of mechanical transduction related protein YAP(Yes-associated Protein)to observe its intracellular distribution,it is found that YAP enters the nucleus after dynamic changes in the Dyn group.The fluorescence staining of nuclear mechanoreceptor protein Lamin A/C showed that its distribution on the nuclear membrane changed uneven.After further treatment with Y-27632 cytoskeleton inhibitor,the expression of osteogenic related genes ALP and RUNX2 and Lamin A/C in Dyn,Dyn+Y27632(Dyn(-))and Sta-F+Y27632(Sta-F(-))clearly shows that both the nucleus and the cytoskeleton play a key role in changing cell fate during dynamic changes.Finally,based on the needs of clinic,we combined the 4D printing technology and programmable hydrogel technology with the shape memory dynamic topology interface to construct a bilayer dynamic membrane with a personalized shape.The upper programmable layer is prepared by mixing Ethyl acrylate(HEA),PCLDA(Mn=2000),and 3-Sulfopropyl methacrylate potassium salt(PSPMA).The hydrogel can achieve2.5-12 times swelling to drive the bilayer curl.Through digital light processing(DLP)to control the exposure time of light,it is found that when the bilayer membrane is cross-linked under 25-80 s light,its shape memory layer has a programmable inner diameter of 2.13-5.49mm(0.4mm),3.32-5.78mm(0.5mm)and 3.88-6.88mm(0.6mm).And through the regional cross-linked hydrogel,a complex and controllable macroscopic structure is constructed after swelling.Furthermore,observation of the microstructure of the SMP layer through CLSM shows that the system can achieve X dynamic changes on the curling surface.Subsequently,C57 mice were used to construct a fracture defect model,and the bilayer dynamic group(Dyn-Bilayer),the bilayer static group(Dyn-Sta-F)and a blank group(Control)were established.Micro-CT results showed that the Dyn-Bilayer group achieved bone healing at 4 weeks while the other two groups were still in the repair process.Calculating bone volume fraction(Bone Volume/Tissue Volume,BV/TV)shows Bilayer-Dyn> Bilayer-Sta-F> Control.Morphological analysis of tissue sections and immunofluorescence staining sections was used to evaluate the bone healing state.Staining of RUNX2,OSX and CD34,showed that there were more related cell existed around the bone defect in the Dyn-Bilayer group,indicating that this group could better promote bone healing.Conclusion1.The shape memory polymer modified based on PCL has suitable biocompatibility,stimulus response temperature and shape memory recovery ability.2.By manipulating the dynamic interface,the adhesion and proliferation of the cells can be accelerated in the early stage,and the morphological change in the later stage promotes the osteogenic differentiation of the cells and realizes more formation of new bone.3.The shape memory dynamic topological interface promotes the osteogenic differentiation of bone marrow mesenchymal stem cells through cytoskeleton stretching and nuclear deformation.4.Through the combination of programmable hydrogel and DLP technology,a programmable bilayer membrane system is constructed,which realizes segmented control of the personalized construction of the macroscopic shape and the controllable change of the microscopic shape.The bilayer dynamic membrane can accelerate bone repair in mouse femur fractures,promote the osteogenic efficiency and vascularization. |
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