Construction And Biological Evaluation Of Bacterial Cellulose Based Bi-layered Tissue Engineered Urethral Scaffold

Urethral stricture is a common disease after urethral injury,which seriously affects the quality of life of patients.It is often caused by inflammation,trauma,congenital defect,malignant tumor and iatrogenic operation.At present,autologous tissue grafts,such as penile flaps,oral mucosa and bladder mucosa,are mainly used to treat urethral stricture.However,autologous tissue transplantation has some problems,such as"limited donor","complications of donor site"and"secondary operation".With the development of regenerative medicine,tissue engineering technology has provided a new effective method for the treatment of urethral stricture.The urethra is composed of mucosal layer and cavernosum layer.The regeneration of urethra mucosal epithelium and the rapid construction of vascular network are the key to achieve the physiological repair of urethra using tissue engineering technology.In this paper,inspired by urethral tissue structure,porous scaffolds with directional channels and Sodium alginate based hydrogel were used to construct bi-layered biomimetic scaffolds.TEMPO oxidated bacterial cellulose（TOBC）and silk fibroin（SF）were used to prepare directional channel 3D porous scaffolds.The micro-nano structure（micron-scale holes,nanofibrous structure）can not only beneficial for cell ingrowth and proliferation,but also promote the formation of vascular network after implantation.The hydrogel constructed by TOBC and sodium alginate（ALG）which mimetics urethral mucosa can promote epithelialization.The synergism of epithelialization and vascularization promote urethral defect repair.The specific research contents are as follows:（1）SF/TOBC scaffolds with directional channels are prepared by directional freeze drying method.And the morphology,mechanical strength,water absorption and degradation properties of the scaffolds were characterized.The results show that:with the increase of the solid content of SF/TOBC solution,mechanical properties of porous scaffolds significantly increased,the average size of channels decreased.Combined with the mechanical properties and the channel size factors,we chose the solid content of 3%SF/TOBC to prepare scaffolds for the follow-up studies.With the increase of TOBC content in porous scaffolds,the pore size,mechanical strength and water absorption performance of scaffolds showed increasing trend,while the degradation rate of scaffolds would slow down.When the mass ratio of SF to TOBC was 1:1,the average channel size of SB1scaffold was 77μm.Cell experiments in vitro showed that the scaffold could guide cell arrangement and migration,and was more conducive to the adhesion,proliferation and growth of HUVECs.（2）Alginate hydrogels were prepared using calcium carbonate-gluconolactone（Ca CO₃-GDL）system.TEMPO-oxidized bacterial cellulose was introduced as enhancement nanofibers and can mimic the collagen nanofiber structure in natural extracellular matrix（ECM）.The microstructure and stiffness of the hydrogel were regulated by changing the concentration of Ca²⁺in the crosslinking system,and the conductivity of the hydrogel was enhanced by adding PEDOT:PSS.The results showed that with the increase of Ca²⁺concentration,the network structure of hydrogels shrank,and the pore size decreased,while the surface of hydrogels with TOBC was coarser than that of pure sodium alginate hydrogels.With the addition of TOBC and the increase of Ca²⁺concentration,the mechanical properties of hydrogels were enhanced and the equilibrium swelling rate of hydrogels decreased.The higher the ratio of PEDOT:PSS in hydrogels,the better the conductivity.After H₂SO₄treatment,many linear"strip"structures appeared on the surface of SA/TOBC/PEDOT hydrogels,and the conductivity increased from 0.47 S/m to 1.94 S/m.In vitro cell experiments showed that hydrogel with higher stiffness could promote the migration of SV-HUC-1,and hydrogel with higher electrical conductivity promoted cell proliferation and viability.