Adipose-derived Stem Cell Sheets Combined With Polyglycolic Acid For Bladder Reconstruction

Objective(1)Adipose-derived stem cells(ADSCs)were isolated and cultured in vitro.The multiple differentiation potential of ADSCs were performed.Then,we evaluated the feasibility of ADSCs as a source of seeding cells for producing the tissueengineered bladder.(2)The super-paramagnetic iron oxide(USPIO)nanoparticles were prepared,and the characterization of USPIO was detected.The labeling efficiency,cytotoxicity and MRI signal intensity of USPIO-labeled ADSCs were analyzed.(3)Vitamin C induced USPIO-labeled ADSCs to prepare ADSC sheet.we observed the ultrastructure of cell sheet and established the method of constructing tissueengineered bladder patch with ADSC sheet and polyglycolic acid(PGA)scaffolds.(4)MRI was performed to dynamically monitor the ADSC sheet at the site of bladder reconstruction.Histological and urodynamic testing were performed to analyze the therapeutic effect of tissue-engineered bladder patch on bladder reconstruction.The tissue-engineered bladder patch provides a new therapeutic strategy for the construction of the engineered neobladder in the future.Methods(1)ADSCs were isolated from the inguinal fat pad.Fresh adipose tissues were digested with collagenase type I to obtain ADSCs.To identify the multiple differentiation potential,ADSCs were induced to differentiate by switching to osteogenic,adipogenic or myogenic differentiation medium.Oil Red O staining,Alizarin Red staining and immunohistochemical staining of desmin were respectively performed to evaluate the extent of adipocyte,osteoblast,and myoblast formation.(2)USPIO were synthesized by hydrothermal method.The microstructure and size of the synthesized USPIO were characterized using transmission electron microscopy(TEM).The crystalline structure of the synthesized USPIO were analyzed by an Xray diffractometer(XRD).The magnetization loop of USPIO were measured by The Physical Property Measurement System(PPMS).(3)Different concentrations of USPIO were used for labeling ADSCs,Prussian blue staining was used to analyze labeling efficiency.Cytotoxic test was used to determine the safe range of USPIO labeling concentration.MRI imaging was used to determine the concentration of USPIO required to label ADSCs and cause a sufficient decrease in MRI signal intensity.The best USPIO labeling concentration was determined based on the above results.The safety of the optimal concentration of USPIO was further analyzed by flow cytometric detection of cell cycling and apoptosis.The distribution of USPIO in subcellular compartments was observed by TEM imaging of USPIO-labeled ADSCs.(4)To create a cell sheet,the ADSCs labeled with USPIO were plated into a temperature-responsive cell culture dish.The ADSC sheet was constructed by vitamin C induction.The surface morphology of the ADSC sheet was observed under scanning electron microscopy(SEM).The tissue structure of ASC sheets was evaluated by hematoxylin and eosin(HE)staining,Masson's trichrome staining and Prussian blue staining.(5)The cylindrical PGA scaffolds were prepared,and the tissue-engineered bladder patch was constructed by the combination of multi-layer USPIO-labeled ADSC sheet and PGA scaffolds.The biocompatibility between the ADSC sheet and the PGA scaffolds was observed under scanning electron microscopy.(6)The tissue-engineered bladder patch was evaluated in a bladder augmentation model using female SD rats.MRI was performed to monitor the USPIO-labeled ADSC sheet after implantation.Urodynamic studies were performed to evaluate the bladder function.Hematoxylin and eosin(HE)staining and Immunofluorescence staining were performed to examine the histological structure of the engineered neobladder.Results(1)Primary cultured ADSCs generated from fresh rat adipose tissue proliferated rapidly and cultured as a monolayer.ADSCs began to be attached after 24 h and reached70-80% confluence within 5 days.Under appropriate induced culturing conditions,adipogenic differentiation was confirmed by the formation of lipid droplet indicated by the Oil Red staining.Osteogenic differentiation was demonstrated by the deposition of mineralized matrix detected by Alizarin Red staining.In addition,myogenic differentiation was observed as demonstrated by the expression of myogenic marker desmin.(2)TEM images showed that the USPIO nanoparticles were approximately spherical and fell within a relatively narrow diameter range of 2–11 nm,which has good dispersion.The calculated diameter of the USPIO was 8.02 nm,based on the diffraction peak(311)observed in XRD,which is consistent with the image result of TEM.The hysteresis curve shows that the synthesized USPIO has superparamagnetic properties.(3)Prussian blue staining showed that the efficiency of USPIO-labeled ADSCs increased as the USPIO concentration was increased.The cytotoxicity of the USPIO was analyzed using a standard CCK-8 assay,which showed that cytotoxicity was observed at the USPIO concentrations of 50 ?g/ml.The reduced MRI signal could be easily observed by the naked eye when the concentration of USPIO reached greater than or equal to 25 ?g/ml.Hence,25 ?g/ml was selected as a safe and effective concentration of USPIO to label ADSCs.These results of cell cycling and apoptosis further confirmed that 25 ?g/ml USPIO did not affect cell viability.The USPIO particles were predominantly existed in the endosomes/lysosomes.(4)The sheet of ADSCs was formed after 14 days of culture in the induced medium.Scanning electron microscopy showed that that the ADSC sheets contained multilayered cells and abundant extracellular matrix(ECM).HE staining showed that the ADSC sheet was composed of five or six layers of ADSCs.Masson's trichrome staining showed that a large amount of collagen fibers was observed outside the ADSCs.Prussian blue staining showed a lot of blue-stained USPIOlabeled ADSCs distributed in the cell sheets.(5)PGA was used to make cylindrical scaffolds.USPIO-labeled ADSC sheets were transferred onto the highly porous PGA scaffolds to construct a tissue-engineered bladder patch.The USPIO-labeled ADSC sheets adhered to the PGA scaffolds after transfer.Under scanning electron microscopy,it was found that USPIO-labeled ADSC sheet rich in cells and extracellular matrix was evenly and tightly attached to the surface of PGA scaffolds.(6)The USPIO-labeled ADSC sheets could be dynamically visualized by MRI.Relative hypointensity representing USPIO-labeled ADSC sheets was observed at the transplantation site of bladder.At 8 weeks after transplantation of tissue-engineered bladder patch,the regeneration of urothelium,smooth muscle,blood vessels and neural cells could be seen at implanted sites.Bladder capacity and bladder compliance were assessed in an urodynamic detection.Bladder capacity increased continuously after implantation of tissue-engineered bladder patch.Bladder compliance in the bladder patch group nearly recovered to the normal level.Conclusion(1)In this study,the ADSCs isolated from adipose tissue exhibited the potential of adipogenic,osteogenic and myogenic differentiation.(2)The ADSCs were labeled with USPIO at a concentration of 25 ?g/ml,which did not affect cell cycle and apoptosis.No cytotoxicity was observed at the USPIO concentrations of 25 ?g/ml.USPIO-labeled ADSCs could be clearly detected by MRI in vitro.(3)ADSC sheet labeled with USPIO had good compatibility with the PGA scaffold.The tissue-engineered bladder patch was successfully constructed using ADSC sheet and the PGA scaffold.(4)Tissue-engineered bladder patch has better therapeutic effect for reconstructing the bladder.The structure and function of an engineered neobladder was close to that of normal bladder.The USPIO-labeled bladder patch provides a safe and effective therapeutic strategy for bladder regeneration.