Research Of Bone Tissue Engineering Based On Human Urine-derived Stem Cells

OBJECTIVE: The current study investigates the isolation of urine-derived stem cells(USCs) from human clean midstream urine. The biological characteristics of USCs, such as proliferation rate, surface markers and multilineage differentiation potential, were examined and compared with human adipose-derived stem cells(ASCs). USCs were transduced with human bone morphogenetic protein 2(BMP2) gene. The osteogenic potential of transduced USCs were examined. Then, the biological behavior of USCs in beta-Tricalcium Phosphate(β-TCP) was evaluated in vitro. Finally, USCs and β-TCP were implanted in critical size femoral defect of rat and investigated the bone regeneration ability of the composite.METHODS: The first part: 200 ml clean midstream urine samples were obtained from health adult. After the urine samples were centrifuged and washed, special mixed culture medium was added. The proliferation ability and surface markers of USCs was examined with CCK-8 and flow cytometry, respectively. After added with differentiation medium, the osteogenic, chondrogenic and adipogenic differentiation potential of USCs was detected. The second part: Lentiviral vector containing human BMP2 gene was constructed in vitro. USCs were transfected with lentiviral. Gene transduction efficiency and cell viability assay was performed. ALP activity, mineral calcium deposition and protein expression of bone-related markers was carried out to detect the osteogenic potential of lentiviral-BMP2 transduced USCs. The ectopic bone formation ability of lentiviral-BMP2 transduced USCs was also examined in vivo. The third part: USCs were isolated, amplified and seeded into β-TCP. The attachment, survival and proliferation of USCs in β-TCP was detected using scanning electron microscope, live-dead staining and CCK-8, respectively. The ALP activity and expression of osteogenic related genes was detected after 7 and 14 days cultured with osteogenic induction medium. The fourth part: USCs/β-TCP were implanted into rat 6mm femoral defect. The healing of bone defects was examined through X-ray, micro-CT and histological staining at 4, 8 and 12 weeks after surgery.RESULTS: The first part: Cell colonies were observed in the culture plate at 5-7 days after initial plating. The cells colonies reached 90% confluence after 14 days. CCK-8 assay showed that USCs had a robust proliferation capability and displayed typical “S”-shape growth curves, which was similar with ASCs. Flow cytometer analysis showed that both USCs and ASCs were positive for CD29, CD44, CD73, and CD90 antigen, and negative for CD31, CD34, CD45, CD133 and HLA-DR. USCs were weakly positive while ASCs were strongly positive for CD105. USCs possess the ability to differentiate into osteoblast, chondrocytes and adipocyte, which are similar with ASCs. After serial passages in vitro, USCs still displayed a normal karyotype. The second part: The flow cytometer showed the transduction efficiencies were over 90%. Cell proliferation assay showed that transduction had no significant effect on cell viability. Lentiviral-BMP2 transduction significantly enhanced the gene expression and protein excretion of BMP2. ALP activity and mineral calcium deposition in lentiviral-BMP2 transduced groups were significantly higher than that of normal USCs or lentiviral-GFP groups. Lentiviral-BMP2 transduced USCs expressed bone related marker: Runx2 and OCN. Histological staining revealed that large new bone regenerated in lentiviral-BMP2 transduced USCs group. The third part: USCs could adhere, survival and proliferate on β-TCP. The increased ALP activity and high expression levels of bone-related genes indicated USCs differentiated into osteoblasts. The fourth part: X-ray and micro-CT analysis showed that USCs/β-TCP promoted robust new bone formation, which was significant more than β-TCP or blank control group. Histological staining also revealed that large amount of new bone formation in USCs/β-TCP group. Fluorescence microscopy showed that mineral apposition rate in USCs/β-TCP group was significant higher than that of β-TCP or blank control group. Anti-human cell nuclei antibody indicated the survival of USCs in the defect site after 12 weeks implantation. The in vivo experiments show USCs/β-TCP significantly stimulated bone regeneration in the bone defect sites.CONCLUSION: USCs can be isolated from adult clean midstream urine. USCs possess similar biological characteristics with ASCs. BMP2 gene transduction significantly enhances the osteogenic potential of USCs. USCs can adhere, survive, proliferate and differentiate into osteogenic lineage in β-TCP. USCs combine with β-TCP can significant enhance the repair of bone defects. USCs can be used as seed cell for bone tissue engineering.