Migration And Myogenic Differentiation Effected By Controlled Delivery Bilayer Scaffold For Bladder Tissue Engineering

Background:Bladder dysfunction and volume loss for various reasons seriously affect the lifequality of patients.Surgical reconstruction of the bladder is often required to restore bladder function.Autologous intestinal tractis were applied to bladder enlargement or in-situ replacementin clinical.But there are still problems such as large trauma,complex surgery and complications.Some acellular biomaterials and synthetic materialsis used to experimental bladder reconstruction,while the long-term effect is unsatisfied.The ideal artificial bladder not only can store urine and protect the abdominal cavity from corrosion,but sufficient regeneration of the bladder muscle layer with complete structure to promote the recovery of the mulriple function of bladder storage and excretion.At present,the research of urinary tissue engineering on the regeneration of bladder muscle layer is madequate.Objective:Try to fabricated a 3D macroporous mesh-electrospinning microporous film core-shell bilayer scaffold which can release heparin stably and continuously.To induce adipose derived stem cells(ADSCs)differentiate into functional smooth muscle cells with the assistance of scaffold.To investigate the feasibility of repairing the bladder defect of rats and improving the function with the scaffold and induced ADSCs.Methods:(1)Firstly,piezoelectric 3D printing was used to prepare mesh-structured supports for macropores.Then the mixed solution of polycaprolactone(PCL)containing bladder acellular matrix(BAM)was used as the outer phase of the shell,and the heparin solution was used as the inner phase.A layer of nuclear spinning fiber film was prepared on the surface of the 3D printed scaffold by coaxial electrospinning technology,and then a two-layer scaffold(PB-HCS/PCL)was obtained.Subsequently,the inner structure of electrospun fibers was observed by transmission electron microscopy(TEM),and the thickness,layers and size of the PB-HCS/PCL mesh bilayer structure were observed by SEM.The sustained release concentration and cumulative release rate of scaffolds(PBH-Mix,P-HCS,PB-HCS)were determined in vitro.(2)ADSCs are extracted from the rat groin area,identified by flow cytometry instrument,then cultured in the mesh,at different time points CCK8 kit was used to detect cell proliferation.ADSCs were cultured in smooth muscle induction medium,no induction medium and PB-HCS/PCLbilayer scaffold plus induction medium combination for two weeks.The cells were stained with rhodamine labeled phalloidin to observe cell migration distribution and morphological changes.Real-time RT-PCR was used to detect the gene expression of ADSCs differentiated into induced smooth muscle cells(AD-iSMCs)in the transcription level.(3)18 rats with partial bladder wall resection were randomly divided into 3 groups,and the bladder defect repair was performed in different ways,including the direct suture group(n=6),the BAM direct repair group(n=6),the PB-HCS/PCL(AICSP)bilayer scaffold repair group(n=6)and the native control group(n=4).Cystography was performed 1 month after the operation,and the bladder tissues were obtained after the rats were sacrificed for pathological examination to observe the hyperplasia of smooth muscle and collagen.Results:(1)The obtained PB-HCS/PCL mesh scaffold exibited a two-layer structure with a membrane-like layer and a large-pore mesh layer.Under the electron microscope,the electrospun membrane layer was composed of nano-sized micropores and fibers with core-shell structure.Through the two-week release experiment,it was found that the structure could continuously release heparin.(2)The mesenchymal stem cell phenotype of extracted rat fat stem cells was identified.Performance showed by the F-actin stainingcell growth along the stent wall,and gradually merged into cell layer structure,the real time RT-PCR manifest that under the synergistic effect ofheparin-BAM and 3D environment,ADSCs gene phenotype transformed tocontractility smooth musclespecific phenotype which conducive to the function of bladder smooth musclerecovery.(3)in vivo tests,defect repair of the bladder after 1 months,urethral retrograde imaging showed each group had no obvious leak.Bladder capacity in direct suture group keep low level,Cystographic area in BAM directly group and AD-iSMCs seeded PB-HCS/PCL mesh bilayer scaffold(AICSP)group were increased,but the BAM group shows signs of overfilling;AICSP group were closer to the normal group.the bladder biopsy showed that both epithelial and muscular layer were regenerated.Muscle bundle of AICSP group were more regular arrangement compared with other groups,obvious new blood vessels were observed.While there were a large number of irregular collagen regeneration and muscle layers form quantity was poor in BAM group.Conclusion:(1)the macropores of mesh in the PBHCS/PCL bilayer scaffold is conducive to the cell growth and migrate deep along the inner wall of the scaffold,and the electrospinning fiber layer in the scaffold can sustainably release heparin,which helps the ADSCs differentiate into genes close to the contraction phenotype smooth muscle cells,and secrete extracellular matrix components similar to normal tissues.(2)the bilayer AICSP scaffold could effectively repair the bladder defect,and the bladder volume was basically normal after reconstruction for 1 month.Meanwhile,the regeneration of the urothelium and muscle layer of the bladder was observed.