Research Of Antibody-modified Bacterial Cellulose Scaffolds For Capturing USCs In Artificial Urine

Objective Urine-derived stem cells(USCs)are considered to be mesenchymal stem cells present in urine,which can be used in combination with biomaterials to reduce complications arising from autologous tissue or acellular matrix transplantation in bladder repair and reconstruction.In this study,a novel CD29 antibody immobilized bacterial cellulose scaffold(anti-CD29 /BCSs)was designed to capture USCs in artificial urine by combining stem cells with materials,providing an experimental basis for clinical bladder tissue repair.Methods1.Stent preparation,characterization and properties testing.Heparin(Hep)and type I collagen(Col)were immobilized on the surface of bacterial cellulose(BC)by layer self-assembly and glutaraldehyde cross-linking techniques to produce anti-CD29/BCSs.The deposition effect of Hep and Col,antibody immobilization efficiency and distribution state were measured by Fourier transform infrared spectroscopy(FTIR),X-ray electron spectroscopy(XPS)and FITC-laser confocal microscopy.The physicochemical properties of anti-CD29/BCSs,including swelling rate,degradability,mechanical strength,and fouling resistance,were tested in artificial urine.2.USCs isolation,identification and cell viability assay.USCs were isolated from human urine samples and cultured using human urine-derived mesenchymal stem cell expansion medium.Surface-specific protein expression of USCs was detected by flow cytometry.The live/dead ratio of USCs in artificial urine at different times was observed by calcein-AM/propidium iodide double staining.3.Assay of scaffold on cell capture capacity in artificial urine.The USC capture ability of anti-CD29/BCSs under artificial urine was detected by rhodamine-ghost pen cyclic peptide with 4’,6-diamidino-2-phenylindole cell staining,and Image J statistics of cell density,spreading area and elongation rate.4.Statistical analysis.Graphpad Prism 8 software was used for statistical and graphical analysis of the obtained data,and one-way ANOVA and Tukey test were performed to analyze the differences between the groups.Results1.FTIR and XPS proved that the prepared scaffolds contained all the typical characteristic peaks of BC,Hep and Col,and with the increase of the number of alternate precipitations,the area covered by Col increased sequentially.The FITC-laser confocal microscopy showed that CD29 antibody was evenly distributed on the surface of anti-CD29/BCSs,and there was no significant difference in the immobilization efficiency under artificial urine flushing.The prepared scaffold has good swelling rate,suitable degradation rate,mechanical strength and stain resistance in artificial urine,which can better adapt to the physiological activities of the bladder.2.Rice-like USCs were isolated from fresh human urine samples.The flow cytometry analysis showed the expression of strongly positive surface markers characteristic of MSCs and negative surface markers characteristic of hematopoietic stem cells and endothelial cells.3.The density of USCs captured by anti-CD29/BCSs in artificial urine(anti-CD29/BCSs-U)was significantly higher than that of the BC scaffold(p < 0.001),which was not significantly different compared to anti-CD29/BCSs in cell culture medium(anti-CD29/BCSs-M).In addition,anti-CD29/BCSs-M captured USCs had higher cell elongation compared to that of anti-CD29/BCSs-U.ConclusionBased on the physicochemical characterization,functional measurements and biological evaluation of the capture scaffold,anti-CD29/BCSs can effectively capture USCs in artificial urine.And USCs can still effectively adhere to the scaffold for a certain period of time,which meets the biomaterial properties as an in vivo bladder repair carrier.