Effect Of Electrospun Polyurethane Nanofibers On The Proliferation And Differentiation Of Tendon Derived Stem Cells And Repair Of Tendon Injury

Objective In this study,a new type of electrospun nanofiber membrane was prepared,which simulates the changes in the structure and composition of the natural tendon-bone connection interface.In in vitro experiments,tendon derived stem cells were cultured on the membrane to verify the effect of the scaffold on cell growth and differentiation.In in vivo experiments,the membranes were planted on the surface of the injured rotator cuff and observed the healing of the rotator cuff injury by the membrane.Methods In this study,the electrospinning solution was prepared by dissolving the spinning material and the photothermal material in a solvent.The electrospinning equipment was used to collect the uniaxially oriented nanofiber membrane with a high-speed roller.Next,the photothermal material was excited to generate heat by laser irradiation,so that the nanofibers reached the melting point and welds were generated,and the uniaxially oriented nanofibers were transformed into randomly arranged nanofibers to realize the continuous gradient change of the structure.Then,the fiber membrane with the structural gradient change was placed vertically in the container,and the mineralization solution was gradually added,so that the randomly arranged part at the bottom had a long soaking time,and the uniaxial orientation soaking time at the upper part was short,and gradient mineralization was generated to obtain a shaped patch material.The microstructures of the membrane were observed by SEM and the mineralization content were quantitatively detected by EDX.In in vitro experiments,the tendon derived stem cells were extracted,and proliferation,multiple differentiation capabilities,and specific protein identification were performed.Then,the tendon derived stem cells were planted on the membranes,and the fiber membrane was used to observe the effect of the fiber membrane on the cell by CCK8 and fluorescent staining.In animal experiments,the gradient nanofiber membranes were transplanted into the rabbit rotator cuff partial injury animal model in situ.After 3 and 6 weeks,HE,Masson,Sirius red staining and tensile test were used to observe the effect of membranes about the healing of tendon to bone.Results In this study,nanofiber membranes with gradient changes in structure and composition were successfully prepared,and the gradient changes in quantitative mineralization content were observation by SEM and detected by EDX.The tensile test of the nanofiber membranes proved that the longitudinal limit mechanical stress of the oriented nanofiber structure was reduced by the temperature influence,and the longitudinal mechanical stress of the nanofiber membrane was improved after the surface mineralization treatment.the extracted tendon derived stem cells were planted on the nanofiber membranes,and CCK8 test confirmed that the nanofiber membrane had no inhibitory effect on the growth and proliferation of tendon derived stem cells.The tendon derived stem cells were fixed after being planted for 3 days,then F-actin/DAPI was used to perform fluorescent staining to observe the cell morphology.It was found that the cell morphology changed with the position of the nanofiber membrane where the cell was located.In the uniaxially oriented part,the cells were more slender and regular.While in the random structure part,the cells arrangement were irregular.The length and width of the cells were concerned.Comparison of the ratio,it was found that the aspect ratio of the cell changes with the change of the structure regardless of whether it was mineralized or not.After 14 days of cell fixation,the cells were stained with Tenascin C/Runx2/DAPI immunofluorescence.It was found that the uniaxially oriented nanofibers induced tendon derived stem cells to express more tendon-associated protein Tenascin C,while in the random structure,the nanofiber structure induces tendon derived stem cells to express more osteogenic related protein Runx2.In vivo experiments,the staining experiments showed that the nanofiber membrane had a promoting effect on the healing of part of the rotator cuff injury.The membrane could promote tissue proliferation and regular arrangement.At the same time,the tensile test showed that the patch group had a higher limit mechanical stress.Conclusion In this study,nanofibrous scaffolds with nanofibrous structure and mineralization gradient changes were successfully prepared.In in vitro experiments,the fibrous scaffolds had no effect on tendon stem cell proliferation.The oriented part of the fibrous membrane induced regular cell arrangement and tendonogenic differentiation,while the random mineralized part induced irregular cell growth and differentiation towards osteogenesis.In in vivo experiments,it was observed that the membranes promoted the repair of the injured rotator cuff and the formation of type I collagen.It also improved the final tension of the injured tendon.We believed that this method can be extended to the production of three-dimensional scaffolds,providing a new method for full-thickness rupture of tendons or repair of tendon defects.