Co-delivery Of VEGF And BFGF Via A PLGA Nanoparticle-modified Bam For Effective Contracture Inhibition Of Regenerated Bladder Tissue

Varieties of congenital and acquired bladder anomalies and diseases,such as neurogenic bladder,bladder exstrophy,or posterior urethral valves,may require augmentation cystoplasty.The current primary standard of surgical treatment is bladder replacement or augmentation with gastrointestinal segments.However,the substitution of intestinal tissue within the urinary tract is fraught with many potential complications,such as urolithiasis,infection,metabolic abnormalities and even malignancy.Bladder acellular matrix(BAM)has excellent histocompatibility,which is considered to be an ideal and optimal substitute.However,the pure degradable scaffold alone does not provide a satisfactory result because it can elicit various negative effects,such as fibroblast depiction,collagen deposition,scar formation,and grafting contracture,over time.The early and rapid building of a mature vascular network is critical to facilitate grafting of organoids and to support long-term tissue survival.Many incorporation methods,such as injection,rehydration,and incubation,have been demonstrated to enhance the ability of the BAM to mediate bladder tissue regeneration in the short term(2 or 3 weeks).However,the introduced growth factors may not have a noticeable effect for a long time.Many strategies for incorporating exogenous growth factors into the BAM,such as rehydration,repeated lyophilisation and rehydration,or injection and incubation,have enhanced the ability of the BAM to mediate bladder tissue regeneration by promoting angiogenesis,muscular growth and inhibiting graft shrinkage.However,these strategies may be effective short term(2-3 weeks),but they do not last a long time after grafting.Nanotechnology is a newly developing drug-releasing technology in the last few years.Nanoparticles-modified BAM may be expected to inhibit efficiently graft contracture and improve the outcome of bladder regeneration.In this paper,we used a poly(lactic-co-glycolic acid)(PLGA)nanoparticle(NP)-modified BAM to co-deliver VEGF and bFGF in an effort to rapidly restore vascular networks and to effectively inhibit contracture in augmented bladders.The main work is as follows:(1)The BAM was prepared by a combination of physical,chemical and enzymatic methods.The morphology of the BAM was observed by digital camera and scanning electron microscope.The results showed that the decellularized BAM appeared to be translucent and smooth with the strength and resiliency,which contribute to its application to bladder replacement.(2)Nanoparticles containing VEGF or bFGF were fabricated by using a double emulsion solvent evaporation technique.Particle diameter was measured by dynamic light scattering(DLS).SEM images were obtained for the assessment of particle shape and surface morphology.The release of VEGF or bFGF was measured in a buffer solution using a PLGA NP-embedded thermo-sensitive hydrogel.The results showed that approximately 60% and 70% of the loaded VEGF and bFGF,respectively,were released from the gel embedded-NPs in the BAM over 60 days.The release profiles of the two proteins exhibited similar patterns,demonstrating that the controlled dual delivery of VEGF and bFGF can be achieved by the hydrogel-entrapped drug NP system.(3)To investigate the cytotoxicity of the NP scaffold on human umbilical vein endothelial cells(HUVECs),the lactate dehydrogenase(LDH)assay was carried out;the results indicated that these NPs induced no toxic effects on the cells.Cell viability and proliferation were also examined.The CCK-8 assay results demonstrated that cells in all the drug-loaded groups(i.e.,VEGF + bFGF,VEGF,and bFGF)displayed significantly higher cell viability than did those in the non-factor groups(i.e.,NPs and BAM)after 24,48 and 72 h.According to the western blot results,the expression levels of proliferating cell nuclear antigen(PCNA)protein in the groups treated with VEGF and bFGF were significantly higher than were those in non-factor groups.Altogether,these results indicate that the drug-loaded NP scaffold may be a good candidate bioactive factor delivery system and for bladder tissue engineering applications.(4)The modified scaffolds were implanted in the bladders of rabbits for up to 12 weeks.The implanted scaffolds were analysed for graft contracture,host cell infiltration,vascularization,collagen degradation and deposition,and regenerated smooth muscle strip contractility.The results indicated that the functional composite scaffold continuously and effectively released VEGF and bFGF and promoted bladder reconstruction with a significant decrease in graft contracture.In addition,the number and arrangement of regenerated urothelial cells and smooth muscle cells as well as microvascular density and maturity were improved in the VEGF/bFGF nanoparticle group compared with the single factor VEGF or bFGF nanoparticle group and BAM alone.