Objective To evaluate the influence of PKH26 labeling on the biological function of the goat nucleus pulposus cells and the biological function of seeded cells in nude mice by in vivo imaging techonology. Methods Primary nucleus pulposus cells were isolated by enzymatic digestion from the nucleus pulposus tissue of the 1-year-old goat disc. The nucleus pulposus cells at passage 1 were labeled with PKH26 and the fluorescent intensity was observed under the fluorescence microscopy. The labeled cells were stained with toluidine blue and collagen type II immunocytochemistry. The cells viability and proliferation characteristics were assessed by trypan blue staining and MTT assay, respectively. Real-time fluorescent quantitative PCR was used to detect the gene expressions of collagen types I and II, and aggrecan. The fluorescent intensity and scope of the nucleus pulposus cells-scaffold composite in vivo for 6 weeks after implanting into 5 6-week-old male nude mice were measured by in vivo imaging technology. Results Primary nucleus pulposus cells were ovoid in cell shape, showing cluster growth, and the cells at passage 1 showed chondrocyte-like morphology under the inverted phase contrast microscope. The results of toluidine blue and collagen type II immunocytochemistry staining for nucleus pulposus cells at passage 1 were positive. The fluorescent intensity was even after labeling, and the cell viability was more than 95% before and after PKH26 labeling. There was no significant difference in cell growth curve between before and after labeling (P gt; 0.05). The real-time fluorescent quantitative PCR showed that there was no significant difference in gene expressions of collagen types I and II, and aggrecan between before and after labeling (P gt; 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology. Conclusion The PKH26 labeling has no effect on the activity, proliferation, and cell phenotype gene expression of the nucleus pulposus cells. A combination of PKH26 labeling and in vivo imaging technology can track the biological behavior of the cells in vivo.
Objective To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro. Methods The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensitywere observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. Thecharacteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkal ine phosphatase (ALP) staining, Al izarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell prol iferation. Results The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before lebel ing and after lebel ing. After osteogenic induction of labeled BMSCs, ALP staining and Al izarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, l ipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS. Conclusion Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal abil ity and multipotent differentiation abil ity; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
Objective To explore the feasibil ity of using PKH26 as a cell tracer to construct tissue engineered bone. Methods BMSCs isolated from the bone marrow of 1-week-old New Zealand white rabbit were cultured. The BMSCs at passage 3 were labeled with PKH26 and were observed under fluorescence microscope. The percentage of the labeled cells wasdetected by Flow cytometer. The labeled cells were induced to differentiate into osteoblasts in vitro and the morphology of the cells after induction was observed under inverted phase contrast microscope. The osteogenic induction was evaluated by ALP staining and Alizarin red staining. The cells labeled with PKH26 were seeded on the bio-derived bone to construct tissue engineered bone in vitro. Then the compound of cells and material were observed under fluorescence microscope. The compound of labeled cells and material were implanted into the rabbit thigh muscle, and the transformation of the labeled cells was observed by fluorescence microscope 14 and 28 days later. Results Fluorescence microscope observation: the BMSCs labeled by PKH26 were spherical and presented with red and uniform-distributed fluorescence, and the contour of the cells were clearly observed when they were adherent 24 hours after culture. Flow cytometric detection revealed that the percentage of labeled cells was 97.2%. After osteogenic induction, the morphology of the cells changed from long-fusiform to polygon-shape or cube-shape, more ECM was secreted, andthe ALP and the Alizarin red staining were positive. At 48 hours after culturing the PKH26 labeled BMSCs with bio-derived bone, the fluorescence microscope observation showed that there was red fluorescence on the surface and inside of the material. At 14 days after implantation, the labeled cells with red and l ight fluorescence were evident in the implantation area; while at 28 days, the cells with red fluorescence were still evident but less in quantity and weaker in fluorescence strength. Conclusion PKH26 can be used as BMSCs label for the construction of tissue engineered bone in vitro and the short-term tracing in vivo.