Research On Tissue Engineered Bladder Based On Biomaterials And Adipose-Derived Mesenchymal Stem Cells

Objective: Congenital and acquired bladder diseases(such as bladder exstrophy,neurogenic bladder,malignant tumors and trauma,etc.)may cause anatomical or functional defects of the bladder,and surgery is often required to protect kidney and bladder function.Cystectomy and urinary diversion are required,which seriously affects the patient’s quality of life.Enterocystoplasty after cystectomy is the gold standard for bladder reconstruction,but it is often accompanied by a series of complications,such as long-term bacteriuria,stone formation,fibrosis,electrolyte imbalance,anastomotic stenosis,and malignancy.The development of tissue engineering technology provides a new opportunity for the repair and reconstruction of bladder defect.In this study,natural bladder acellular matrix(BAM)and silk fibroin were used as scaffold materials,and adipose-derived mesenchymal stem cells(ASCs)were used as seed cells to construct tissue-engineered bladder.To explore the effect of tissue-engineered bladder prepared with different scaffold materials in the repair and replacement of bladder defect,and to lay a theoretical foundation for the clinical application of bladder repair and replacement.Methods: Using a self-designed bladder perfusion decellularization system,four different perfusion decellularization protocols(group A,group B,group C and group D)were formulated to prepare BAM according to the flow direction of the perfusate and the action time of different decellularization solutions.Compared with the traditional immersion method to prepare BAM for control(group E).The urethral orifice of the bladder tissue was used as the outflow tract of the perfusate in both groups A and B.In groups C and D,part of the tissue at the top of the bladder was cut off,and the opening on the top of the bladder was used as the outflow tract for the perfusate.Groups A and C: the samples were treated with 1% Triton X-100 for 6 h,perfused with deionized water for 1 h,and then treated with 1% sodium dodecyl sulfate(SDS)for 2 h.Groups B and D: the samples were treated with 1% Triton X-100 for 7 h,perfused with deionized water for 1 h,and then treated with 1% SDS for 1 h.Through histological staining and quantitative analysis of bioactive components,a fast and efficient perfusion decellularization protocol was screened.A bi-layer silk fibroin skeleton(BSFS)composed of silk fibroin film and silk fibroin sponge was prepared by extracting silk fibroin aqueous solution from Bombyx mori cocoons.Bladder acellular matrix hydrogel(BAM hydrogel,BAMH)was prepared by pepsin digestion of BAM obtained from perfusion decellularized group C.The rat ASCs were isolated and cultured,and ASCs surface markers(CD29,CD90,CD45,CD106)were identified by flow cytometry.In addition,the ASCs were labeled with CM-Di I fluorescent dye.Live/dead staining and CCK-8 detection were used to evaluate the biocompatibility of the BAM prepared by perfusion decellularized group C and the constructed BSFS-BAMH.Tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs)were jointly constructed with the BAM prepared by perfusion decellularized group C and constructed BSFS-BAMH as scaffold materials,and the CM-Di I labeled ASCs as seed cells.Histological staining and scanning electron microscopy were used to observe the growth and distribution of the ASCs on the BAM and BSFS-BAMH.The constructed tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs),BAM and BSFS-BAMH were embedded in the omentum to promote vascularization,and the histological staining was used to evaluate the vascularization of tissue-engineered bladder patches.Omentum-embedded tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs),BAM and BSFS-BAMH were used to repair the replacement bladder defect,and the cystotomy control group was anastomosis of the excised bladder tissue to the bladder defect.At 2 w,4 w,and 12 w after the operation,the gross morphology of bladder tissue and cystography were performed to observe the morphological recovery of the bladder,and the tissue regeneration of the bladder wall was observed by histological staining.At 12 w after the operation,the urodynamic examination was performed.to assess the recovery of bladder physiological function.Results: H&E and DAPI staining showed that the BAM prepared by perfusion decellularization group C had no obvious nuclear residues.MTS and Alcian Blue staining showed that the collagen structure and glycosaminoglycan(GAG)of the BAM prepared by perfusion decellularization group C were well preserved.The DNA content of the BAM prepared by perfusion decellularization group C was significantly lower than that of the normal group and the BAM prepared by the other four groups.The bladder wall thickness,collagen content and GAG content of the BAM prepared by perfusion decellularization group C were not significantly different from those in the normal bladder group.Scanning electron microscope results showed that a large number of pore structures could be observed on the surface of the BAM prepared by different perfusion decellularization groups,while the pore structure of the BAM prepared by group E was sparse and the pore size was small.The results of gross observation and scanning electron microscope confirmed that the prepared BSFS had good toughness and porous structure,which was beneficial to the adhesion and distribution of seed cells and surgical suture.The prepared BAMH had the characteristics of the hydrogel.When the BAMH was not gelled,it was in a liquid form with fluidity,and after gelation,it was a semi-solid form with a relatively stable structure.The isolated and cultured ASCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29 and the negative expression of CD45 and CD106.Live/dead staining and CCK-8 detection confirmed that the BAM in the perfusion decellularized group C and the constructed BSFS-BAMH had no cytotoxicity.Histological staining and scanning electron microscope observations confirmed that ASCs were widely distributed on the surface and inside of the constructed tissue-engineered patches(BAM-ASCs and BSFS-BAMH-ASCs).The number of regenerated blood vessels in the BAM-ASCs tissue-engineered bladder group was significantly more than that in the BAM group,and the number of regenerated blood vessels in the BSFS-BAMH-ASCs tissue-engineered bladder patch group was not significantly different from the BSFS-BAMH group after omentum wrapped.At 12 weeks after the operation,it was observed that the entire bladder tissue was uniform in shape and the dome-like structure could be observed on the top of the bladder in the tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs)group and the cystotomy control group.The dome-like structure could not be observed on the top of the bladder in the BSFS-BAMH and BSFS-BAMH groups.The continuous urethral layer could be observed in the cystotomy control group and the tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs)group.In addition,the fibrous connective tissue and vascular-like structure could be observed in the lamina propria,and a large number of smooth muscle bundles could be observed in the smooth muscle layer.At 12 weeks after the operation,the bladder capacity,maximum intravesical pressure before voiding,and bladder compliance in the tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs)group were higher than those in the BAM and BSFS-BAMH groups,were lower than those in the cystotomy control group.Conclusion: The self-designed perfusion decellularization system that simulated the physiological process had high decellularization efficiency and short time consumption.Its structure was simple and easy to assemble and build.The BAM prepared by perfusion decellularization group C removed the cellular components that caused immune responses,and well retained the collagen structure and bioactive components that could be used as a scaffold material for bladder tissue engineering.The prepared BSFS was stable in shape,had a porous structure,good flexibility and excellent waterproof performance,and could be used as a skeleton material for building tissue-engineered bladder patch.The prepared BAMH could well simulate the ECM environment of the bladder tissue and provided an ECM "microenvironment" for seed cells.At the same time,the rheological properties of hydrogel could play an anchoring role to make the seed cells distribute in the entire space of the scaffold material evenly.The omentum wrapping could promote the vascularization of tissue-engineered bladder patches.The constructed tissue-engineered bladder patches(BAM-ASCs and BSFS-BAMH-ASCs)could promote the repair of the bladder shape,the regeneration of the bladder wall and the recovery of the physiological function of the bladder,which could provide a theoretical basis for the clinical application of tissue-engineering bladder to repair the bladder defect and accelerate its clinical transformation and application process.