The Tissue Engineering Construction Of Highly Ordered TiO₂Nanotube Arrays Biofiltration Membrane Using The Cell Mixing Implantation Technique

Kidney disease is ranked as the9th major cause of death in the United States byCenters for Disease Control and Prevention. Although conventional renal replacementtherapies （RRT）, such as hemodialysis, hemofiltration and peritoneal dialysis, aresuccessfully used as life-sustaining treatments for chronic renal failure patients, most ofthem still suffer unacceptably high mortality rates. Existing RAD techniques overcome thedefect of RRT, which only replace the functions of gromeruli, but ignore the renal tubularfunctions. In vitro experiments show that RAD can play good tubular function, and hasbeen approved to Ⅰ/Ⅱ clinical trial by FDA. However, the used dialysis membranes arepolymer films, their intrinsic hydrophobicity restrict the application, in addition, they havelow hole density, are unbeneficial for miniaturization and in vivo transplantation.TiO₂nanotube arrays due to its excellent biocompatibility, hydrophily and specialtubular structures, are more and more attractive in biomedical fields. In this study, ordered,open-ended, and high hole density TiO₂nanotube arrays are used as dialysis membranes.However, no work has been reported on the adhesion characteristic of renal cells to TiO₂nanotubes in order to fulfill the kidney functions. Open-ended titania nanotube arrays werefabricated on a pure titanium foil by electrochemical anodization in this group, fourdifferent diameters nanotube materials were acquired via controlled voltage, each materialwas processed by unannealed and without irradiation, unannealed and with irradiation,annealed and without irradiation, annealed and with irradiation, then renal tubularepithelial cells （LLC-PK1） and endothelial cells （ECV304） were seeded on them. Based onit, the effects of illumination performance, crystal structure, and geometry parameters ofTiO₂nanotubes on the adhesion of two cells were studyed detailed. The interaction of cellsand materials was investigated by fluorescence microscope. The cell viability andproliferation were examined by MTT. Simultaneously, the morphologies of LLC-PK1cellsand ECV304cells were observed using filed emission scaning electron microscope（FESEM）. After obtained the optimal parameters for cells growing, the interaction of mixed cells, the adhesion and proliferation effect of collagen and dynamic shear stress ontwo cells were also explored. In addition, the reabsorb function of the new dialysismembrane was preliminary studied.The results showed that the TiO₂nanotube arrays with pore size of about70nm,annealed and without irradiation was optimal adhesion and proliferation for LLC-PK1cells and ECV304cells. FESEM studies showed that the LLC-PK1cells on nanotubearrays exhibited pebbles, and extended filopodia, what’s more, intensive microvilli wereadhered on cell surfaces. ECV304cells had round shapes on nanotubes. Mixture seededcells promoted single cell grown better on nanotubes, and the activity of mixed cells waslarger than that of two single-cell. The further studies showed that collagen had asignificant role for ECV304cells adhesion, but had no effect on LLC-PK1cells and mixedcells. Dynamic shear stress significantly increased the proliferation, made cells distributedmore evenly and formed a single cell layer. Functional tests also indicated that two cellssurvived well on TiO₂nanotube arrays, and had kidney reabsorption function. Theseresults pave the way to develop a newly bioartificial kidney which possesses mixedfunctions.