| The anterior cruciate ligament(ACL)is an important ligament connecting the femur and tibia in the knee joint and has a high incidence of injury,with an annual incidence of 68.6/100,000.However,due to the lack of blood vessels and cells,the ACL is difficult to heal spontaneously after injury and requires surgical reconstruction.Autografts and allografts have limited sources and immune rejection,and artificial ligaments are increasingly being used clinically for reconstruction.The artificial ligaments used clinically are non-degradable artificial ligaments,which show good recovery results in short-term applications,but suffer from difficulties in infiltration of new tissues and poor orientation in long-term use.Specifically,the dense structure of the artificial ligament makes it difficult for collagen fibers to grow into the interior of the artificial ligament,and even after months of time,only the outer layer of its multilayer structure is infiltrated;secondly,due to the lack of orientation induction,even if some collagen fibers successfully infiltrate into the interior of the artificial ligament,it is difficult for them to form an oriented arrangement and orderly tissue,which further leads to the formation of disordered scar tissue and ultimately causes the reconstruction failure.Strategies have been adopted to improve the pro-cellular infiltration and orientation induction properties of artificial ligaments,but there is no multi-effect strategy to improve infiltration and orientation simultaneously.Therefore,in this study,silk(Silk),PGA(Polyglycolic acid)and PCL(Polycaprolactone)were used as raw materials to prepare artificial ligaments with a 5-layer structure by controlling the ratio of yarns with different degradation cycles(micron fiber reinforced layer)and spinning time(nanofiber induced layer)based on woven molding technology and electrostatic spinning technology.A layer-by-layer induced regenerative artificial ligament with a 5-layer structure was produced.The amount of PGA in the weft yarn decreases from the outer layer to the inner layer,which can degrade to produce gradient pores from the outer layer to the inner layer to promote cell/collagen infiltration;each layer of the 5-layer structure has an oriented nanofiber induction layer,which can provide orientation induction cues to the cells/collagen infiltrated inside the artificial ligament while recruiting cells in the surface layer.The main contents of this study include:(1)based on weaving technology,the multi-gradient micron fiber reinforced layer was prepared by adjusting the weft material ratio(silk:PGA 1:1-1:3)and tissue structure(T2/2,T4/2);further combined with electrostatic spinning technology,the micro and nano fiber composite layer was prepared by adjusting the spinning time;finally,the micro and nano fiber composite layer was laminated layer by layer to produce layer-by-layer induced regeneration(2)characterize the structural features of the artificial ligament by microscopic observation and porosity testing,and select the artificial ligament with similar mechanical behavior to the autologous ACL by mechanical tensile testing;(3)evaluate the gradient degradation performance of the artificial ligament by in vitro accelerated degradation experiments,and analyze the degradation behaviour,internal spatial changes and mechanical decay of each component during the degradation process;(4)evaluate the layer-by-layer induced regeneration by cellular experiments(4)to evaluate the infiltration and orientation of cells/collagen inside the layer-by-layer induced regenerated artificial ligaments through cellular and animal experiments.The results and main conclusions of the study are as follows.(1)Based on woven molding technology and electrostatic spinning technology,artificial ligaments with 5-layer structure of layer-by-layer induced regeneration were prepared by regulating the ratio of yarns with different degradation cycles(micron fiber reinforced layer)and spinning time(nanofiber induced layer).(2)The microscopic observation results showed that the artificial ligaments had a uniform and tight structure;the porosity test results showed that the artificial ligaments had a high porosity,and the porosity of the artificial ligaments of both tissue structures was above 50%;the tensile fracture test results showed that the prepared artificial ligaments had excellent mechanical and mechanical properties,and the fracture strengths of the artificial ligaments of two different tissue structures(T2/2 and T4/2)were not significantly different from those of the natural ligaments.The tensile mechanical test results showed that the artificial ligaments prepared had excellent mechanical and mechanical properties,and the elongation at break of the artificial ligaments with two different structures(T2/2 and T4/2)were not significantly different,46.37 ± 1.28 MPa and 48.58 ± 0.19 MPa,respectively,which were better than those of the natural ligaments,but the T4/2 group had lower elongation at break(25.39 ± 1.84%)and higher stiffness(192.09 ± 15.6 N/mm)than the T2/2 group,which were closer to the elongation at break and stiffness of the natural ligaments.Artificial ligaments of T4/2 structure were preferred for subsequent degradation performance and induction evaluation.(3)The degradation performance tests showed that the outer,middle and inner layers of the artificial ligament showed decreasing gradient pores from degradation.After 42 days of degradation,only sporadic PGA fibers could be observed in the innermost layer,while all the remaining layers of PGA were completely degraded,while the silk yarn and PCL nanofibers in each layer did not change significantly.The porosity of the samples gradually increased with increasing degradation time,indicating that the structural design of gradient degradation helped to gradually increase their porosity during the degradation process.During degradation,the mechanical properties of the artificial ligaments decayed and the fracture strength decreased,but still matched the fracture strength of the natural ACL,indicating that the degradation mechanics of the artificial ligaments could match the needs of tissue regeneration.(4)Induction performance tests showed that the gradient degradation and layer-by-layer induction design of layer-by-layer induced regenerative artificial ligaments significantly promoted cell proliferation,infiltration and collagen fiber orientation in vitro and in vivo.In vitro studies showed that the degradation of the gradient pore producing artificial ligament significantly promoted the proliferation of L929 cells compared to the undegraded artificial ligament;in vivo results showed that the layer-by-layer induced regenerative artificial ligament exhibited significant cell/collagen infiltration and orientation with the degradation of PGA. |
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