[Combined Application of Green Fluorescent Protein Labeling and Confocal Laser Scanning Microscope Three-Dimensional Reconstruction to Monitor Construction and in Vivo Transplantation of Tissue Engineered Bone].

OBJECTIVE:The combined application of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods.METHODS:After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was amplified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of liposome. Positive PT67 cells were picked out with G418, and proliferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and proliferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and proliferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts.RESULTS:The isolated cells were fibroblast-like amorphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N 1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The proliferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, proliferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation.CONCLUSION:The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.