Urethral Reconstruction With Modified Tissue Engineered Biomaterial

Part ⅠUrethral Reconstruction with a Gelatin-Bacterial Cellulose Scaffold Seeded with Lingual Keratinocytes in a Rabbit Model Objective: To evaluate the effects of urethral reconstruction with a gelatin-bacterial cellulose(gelatin-BC) scaffold seeded with lingual keratinocytes in a rabbit model.Materials and Methods: Gelatin-BC was prepared and the structure of scaffold was assessedusing scanning electron microscopy. Rabbit lingual keratinocytes were isolated, expanded and seeded onto the scaffold. Gelatin-BCalone(control group, N=10) and gelatin-BC seeded with lingual keratincocytes(experimental group, N=10) were used to repair rabbit urethral defects(2.0×0.8cm). At one and three months post-operatively, retrograde urethrograms and histological analyses were performed to evaluate the outcomes of urethroplasty.Results: The gelatin-BChad a bi-layer structurethat consisted of a compact top surface and an interconnected porous surface. The lingual keratinocytes exhibited good compatibility with the scaffold. At three months, all urethrasmaintained wide calibers in the experimental group.Strictures were found in all rabbits in the control group. Histologically, at one month, intact epithelium occurred in the experimental group, and discontinued epithelium was found in the control group. However, both groups exhibited similar epithelial regeneration at three months. The smooth muscle content and endothelia density in the experimental group were significantly higher than the corresponding values in the control group at bothtime points(p<0.05). Additionally, retrieved urethrasfromthe control group exhibited inflammatory reactions and fibrosis.Conclusion: Our results demonstrated that gelatin-BCseeded with lingual keratinocytes enhanced smooth muscle regeneration and neovascularization without inducing inflammatory reactionsand fibrosis. Gelatin-BCseeded with lingual keratinocytes can potentially be used as a scaffold for urethral reconstruction.Part ⅡReconstruction of Penile Urethra with the 3-Dimensional Porous Bladder Acellular Matrix in a Rabbit ModelObjective: The goal of this study was to evaluate the effect of reconstruction of penile urethra with the 3-dimensional(3-D) porous bladder acellular matrix(BAM) in a rabbit model.Materials and Methods: In 30 male rabbits, a ventral urethral mucosal defect(1.5×0.8 cm) was created. Substitution urethroplasty wasperformed with 5% peracetic acid(PAA)-treated BAM(15 rabbits, PAA-treated BAM group,3-Dporous BAM) and non-PAA-treated BAM(15 rabbits, non-PAA-treated BAM group) in an onlay fashion. At 1, 2 and 3 months after surgery(five rabbits at each time point) in the two groups, retrograde urethrogram and histological analysis were performed to evaluate the outcomes of urethroplasty.Results: In the PAA-treated BAM group, 13 rabbits maintained a wide urethral caliber without a fistula or stricture. In contrast, 10 rabbits kept a wide caliber in the non-PAA-treated BAM group. Histologically, at 1, 2 and 3 months after surgery, the speed of urothelium regeneration in the PAA-treated BAM group was faster than that in the non-PAA-treated BAM group. The smooth muscle to collagen ratio and content of smooth muscle in the PAA-treated BAM group were significantly higher than that in the non-PAA-treated BAM group at each time point(p<0.05). The endothelium density between the non-PAA-treated BAM and PAA-treated BAM groups revealed a significantly increase at all three time points(p<0.05). Conclusion: our results confirmed that the PAA-treated BAM urethroplasty enhancedurothelium, smooth muscle regeneration and neovascularization compared to non-PAA-treated BAM. The 3-D porous BAM as an optimized biological scaffold may be used for cell-based tubular and long-segmental urethral reconstruction in future.Part ⅢTissue Performance of Bladder following Gelatin-Bacterial Cellulose Scaffold and 3-Dimensional Porous Bladder Acellular Matrix implantation in a Rabbit ModelObjective: To investigate the tissue performance of bladder following gelatin-bacterial cellulose scaffold(gelatin-BC)implantation compared to3-Dimensional Porous bladder acellular matrix(3-D porous BAM)in a rabbit model.Materials and methods: Novel gelatin-BC and 3-D porous BAM were prepared, and their porosity and pore size distribution were measured. Gelatin-BC and 3-D porous BAM were separately transplanted into opposite walls of the bladder of 20 rabbits after stripping the bladder mucosa and smooth muscle(1.5×2.0 cm). At4 and 8 weeks post-op, the animals(ten rabbits at each time point) were euthanized. The tissue engineered bladder constructs were then assessed by gross anatomical observation, and histological analysis and muscle contractility studies were performed to evaluate tissue regeneration at both matrix implant sites.Results: The porosity and larger pore size of gelatin-BC were similar to 3-D porous BAM(P>0.05). At 4 weeks, the presence of vesical calculus was evident in 4/10 rabbits.However, at 8 weeks, there was not vesical calculus in rabbits. Histologicalanalysis showed that gelatin-BC and 3-D porous BAM promoted similar degree of urothelium, smooth muscle and blood vessel regeneration(P>0.05).In addition, muscle strips supported by gelatin-BC displayed similar contractile responses to carbachol, KCl, and phenylephrine to 3-D porous BAM. At 4 and 8 weeks, both matrices did not elicit significantacute and chronic inflammatory reactions.Conclusion: Our results demonstrated that gelatin-BC has similar ability to promote bladder tissue regeneration with structural and functional properties to 3-D porous BAM, and with similar biocompatibility. Gelatin-BC and 3-D porous BAM as two optimized biomaterials may have similar effect in urethra reconstruction.