Research On Bioinspired Device For Bone-ligament Interface Of Melt Direct Writing

The integration between bone and ligament is formed through specialized interface tissues called attachment points.The complex and heterogeneous structure of the interface is crucial to ensure a smooth transfer of mechanical stress between bone and soft tissue.The interface is difficult to regenerate after injury,which can easily lead to ligament rupture.Tissue engineering is a promising strategy for interface regeneration.However,the complex structure and cellular composition of native interfaces make interface tissue engineering particularly challenging.Therefore,it is likely that the combination of biomaterials and stimulation of cells with appropriate biochemical and mechanical cues will be required.In recent years,many strategies have been employed to study the bone-ligament interface,which are of great value for both tissue regeneration and understanding the mechanisms of interface regeneration.However,how to generalize the complexity and heterogeneity of the natural bone-ligament interface including structural,cellular,and mechanical gradients is still challenging.Melt Electrospinning Direct Writing(MEW)is an emerging technology capable of precisely depositing micro-nanofibers,and has been widely used in the fabrication of medical devices,especially in bone-ligament interface repair.Therefore,this thesis proposes a new research idea for the construction of the repair device of the bone-ligament interface.In order to solve the trouble of bone-ligament interface repair,this thesis uses melt electrospinning direct-writing technology to print grid-crimp composite micropatterns that simulate the bone-ligament interface,and then a tubular bioinspired device was constructed to show the application prospect of this structure in bone-ligament interface tissue engineering.The research contents is as follows:First,a melt electrospinning direct-write printing device was developed and the fiber deposition rules and stacking consistency of polycaprolactone(PCL)under electrostatic field were explored.Then,a new method of micropatterns with crimped structure was developed,and crimped fiber micpatterns and multi-layer devices with small crimp angle error were prepared.Fiber and device samples were then observed under a scanning electron microscope at an accelerating voltage of 15 k V in EDX mode to study the diameter distribution of grid fiber devices and crimped fiber devices.Fiber diameters and spacing were measured by analyzing SEM images using the Image Pro Plus 6.0 soft imaging system.Results turns out that the new method developed can achieve precise printing effect.Cell orientation experiments with crimped micropatterns were then performed,and for cytoskeletal alignment analysis,cells were stained with 100 n M Phalloidin-TRITC and DAPI for fluorescence imaging using an inverted fluorescence microscope.Cell orientation angles were analyzed using the Image Pro Plus 6.0 soft imaging system.Fibers with different spacing and crimped angles have different induction effects on cell orientation.After that,the process optimization of the grid structure micropattern was carried out,and the grid micropatterns and multi-layer devices with small pore size,uniform diameter and uniform morphology were prepared.Using a test speed of 5 mm/min until the devices was tensile fractured,the results show that the device has mainly linear mechanical properties and bioinspired bone tissue.Then,the osteogenesis experiment of the grid micropatterns was carried out.The quantitative and staining experiments of alkaline phosphatase(ALP),the staining of alizarin red(ARS)and semi-quantitative experiments were carried out on the grid patterns of 50?m,100?m and 200?m pore size.The alkaline phosphatase and calcium nodules expressed by Saos-2 osteosarcoma cells were qualitatively and quantitatively analyzed.The 50?m grid patterns has a more obvious effect of promoting osteogenesis.Finally,the cells co-culture experiment of grid-crimped composite micropatterns was carried out,and the migration behavior of Saos-2 osteosarcoma cells and NIH/3T3 fibroblasts was tracked with Celltracker red and green tracers,The composite micropattern of spacing of 50?m has a better effect of promoting cell aggregation,however,the micropatterns of three fiber spacing had no significant effect on cell proliferation.In addition,a bioinspired tubular device was constructed,and mechanical tensile experiments were performed.The mechanical properties of the bioinspired bone ligament interface device and the native bone ligament interface were similar.All the results indicate that the grid-crimp composite device printed by melt direct writing is a potential graft for reconstruction of the bone-ligament interface.