The Construction Of Bi-layer PLGA/CoⅠ-MeHA Urethral Scaffold For Urethral Reconstruction

Objective:The reconstruction of the injured urethra is still a significant challenge in clinical urology.There are many substitutes for urethral reconstruction,such as autologous tissue and tissue engineering scaffolds,but they are immature in structure and cannot well meet clinical needs.To this end,we designed and prepared a double-layered PLGA/Co I-MeHA urethral tissue engineering scaffold based on the natural urethral structure,and investigated its application in rabbits,and studied its role and effects in urethral reconstruction..Methods:PLGA and Co I were selected as materials,and electrospun films with different PLGA/Co I ratios（7:3/8:2）were prepared by electrospinning.Different ratios of HA and MA were reacted to obtain MeHA with different grafting ratios,and MeHA with suitable grafting ratios was screened by mechanical strength,rheology and in vitro degradation.Rabbit urethral smooth muscle cells and rabbit urethral epithelial cells were isolated and purified by rabbit bladder biopsy,and then loaded with MeHA prepolymer after immunofluorescence staining.Double-layer PLGA/Co I-MeHA urethral scaffold was obtained by using a custom-made tetrafluoroethylene mold,using a PLGA/Co I electrospun membrane as the base,and then adding a MeHA prepolymerization solution loaded with rabbit urethral smooth muscle cells to form a light-curing gel.Forty male New Zealand white rabbits（3-3.5kg,3-4 months of age）were used in animal experiments and divided equally into four groups.Group A:PLGA/Co I-MeHA urethral scaffolds loaded with cells were implanted;Group B:PLGA/Co I-MeHA urethral scaffolds implanted without cells were implanted;Group C: urethrotomy was followed by suturing with autologous tissue（gold standard group）;Group D: urethrotomy was not treated（blank group）.A 1 × 0.5cm defect was created in the rabbit anterior urethra to obtain a rabbit urethral defect model.After the stent was sterilized,it was implanted into the rabbit urethral defect and the urethral repair was observed by retrograde urography three months later.Result:We successfully selected suitable PLGA/Co I electrospun membranes（7:3）and MeHA（40.72%）.The PLGA/Co I electrospun nanofibers have a diameter of144.13±38.22 nm,a porosity of 90.99±3.39%,and a tensile mechanical strength of0.4Mpa,meeting the mechanical strength and suture requirements of animals;they are completely degraded in vitro in about two months.MeHA hydrogel can support the structure and survival of cells with a maximum compressive strength of 38.13 kpa and a degradation time of about 40 days in vitro.The biocompatibility experiments showed that PLGA/Co Ⅰ electrospun membrane and MeHA hydrogel had good biocompatibility.Cell-loaded PLGA/Co I-MeHA urethral stents were implanted into the rabbit urethral defect model.After three months of follow-up,one rabbit died in each group except for the gold standard group.The causes of death were all due to infection.By retrograde urography,it was found that the urethral defect was well repaired in rabbits of groups A and C;three rabbits of group B developed stenosis;and six rabbits of group D developed stenosis,of which four had fistulas.All the results indicate that the PLGA/Co I-MeHA urethral stent can well repair 1 × 0.5cm urethral defect and has great potential for clinical application.Conclusion:In this study,we successfully designed and fabricated a bi-layer PLGA/Co I-MeHA urethral scaffold using PLGA/Co I electrospun membrane and MeHA,which mimics the natural urethra in structure and has good mechanical properties and biocompatibility.The urethral defect was repaired well after in situ implantation of the scaffold in the rabbit urethral defect model for three months,which confirmed the efficacy of the scaffold in the treatment of urethral defect in vivo.It may serve as an effective clinical application to cure urethral defects in human patients.