title: [Application of PKH26 labeling combined with in vivo imaging technology in intervertebral disc tissue engineering].
abstract: 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 > 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 > 0.05). Strong fluorescence in nucleus pulposus cells-scaffold composite was detected and by in vivo imaging technology.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Deapartment of Spinal Surgery, Laboratory of Orthopaedic Institute, Tianjin Hospital, Tianjin, 300211, P.R.China.
authors.forename: Yaohong
authors.initials: Y
authors.lastname: Wu
authors.forename: Baoshan
authors.initials: B
authors.lastname: Xu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yang
authors.forename: Xiulan
authors.initials: X
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xinlong
authors.initials: X
authors.lastname: Ma
authors.forename: Qun
authors.initials: Q
authors.lastname: Xia
authors.forename: Chunqiu
authors.initials: C
authors.lastname: Zhang
authors.forename: Haiwei
authors.initials: H
authors.lastname: Xu
authors.forename: Chao
authors.initials: C
authors.lastname: Zeng
mesh_terms: D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D017690:Cell Transplantation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D006041:Goats; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D061848:Optical Imaging / Q000379:methods; D009930:Organic Chemicals; D013194:Staining and Labeling; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D009930:Organic Chemicals; C070080:PKH 26