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.
Objective To investigate the venous drainage in retrograde island flaps by fluorescence tracing technique and to observe the pathway of venous drainage. Methods The 0.1mL venous blood was collected from the marginal ear vein of every rabbit (n=20), respectively, and erythrocytes were separated by centrifugation and then were labeled with FITC. Positive rate and fluorescence intensity of FITC-labeled RBC were detected by flow cytometry. RBC morphous was observed under the inverted fluorescence microscope. Saphenous retrograde island fasciocutaneous flap and antegrade islandfasciocutaneous flap (4.0 cm × 3.0 cm in size with vascular pedicle length of 3.0 cm) were successfully establ ished in hind l imbs of 20 New Zealand white rabbits．One hind l imb of each rabbit was randomly assigned as the experimental group and the contralateral side was assigned as the control. The same flap was establ ished in the control group without any fluorescence tracer. According to retrograde or antegrade flaps, the experimental group was divided into 2 groups with 10 rabbits in each group. And then, according to different pathways of tracer-giving, each group was divided into 2 subgroups of artery and vein, with 5 rabbits in each subgroup. The labeled erythrocytes (5 μL) were injected into artery or vein and then flaps were cut down 5 seconds later. The flaps were immediately frozen and chipped (5-7 μm). Consecutive three frozen sections were made and two of them were stained with HE and GENMED, respectively, but the third one was squashed without staining. All frozen sections were observed under the microscope. Results Positive rate of FITC-labeled RBC was beyond 99% and fluorescence intensity was more than or equal to 103. FITC-labeled RBC showed steady green fluorescence under the inverted fluorescence microscope. Fluorescence appeared in all experimental groups, but none was found in the control groups. In antegrade island flap group, fluorescence appeared mainly in lumen of vein, wall of vein and inner membrane and outer membrane of artery. In retrograde island flap group, fluorescence distributed principally in inner membrane and outer membrane of artery and wall of vein. Conclusion The fluorescence tracing is appl icable to the research of venous drainage. Venous drainage in the antegrade island flaps is mainly through lumen of vein, wall of vein and inner membrane and outer membrane of artery. While, venous drainage in retrograde island flaps is principally through inner membrane and outer membrane of artery and wall of vein.