title: Surgical removal and controlled trypsinization of the outer annulus fibrosus improves the bioactivity of the nucleus pulposus in a disc bioreactor culture.
abstract: BACKGROUND
  The maintenance of nucleus pulposus (NP) viability in vitro is difficult. The annulus fibrosus (AF) pathway reflects one nutrient transport channel and may have an important effect on NP viability in disc organ cultures. The present study describes a feasible disc pre-treatment involving the AF and investigates its efficacy in improving NP bioactivity in an in vitro disc bioreactor culture.
  
  
  METHODS
  Rabbit discs that were randomly assigned to the experimental group (EG) were pretreated via the surgical removal and controlled trypsinization of the outer AF. The discs in the control group (CG) did not receive any special treatment. All discs were organ-cultured in a self-developed bioreactor. Solute transport into the central NP was measured using a methylene blue solution. On days 7 and 14, histological properties, cell viability, cell membrane damage, gene expression and matrix composition within the NP in these two groups were compared with each other and with the corresponding parameters of fresh NP samples. Additionally, the structures of the outer AF and the cartilage endplate (CEP) following pre-treatment were also assessed.
  
  
  RESULTS
  The outer AF in the EG became disorganized, but no specific changes occurred in the CEP or the inner AF following pre-treatment. The discs in the EG exhibited increased penetration of methylene blue into the central NP. On days 7 and 14, the NP bioactivity in the EG was improved compared with that of the CG in terms of cell viability, cell membrane damage, gene expression profile and matrix synthesis. Moreover, cell viability and matrix synthesis parameters in the EG were more similar to those of fresh samples than they were to the same parameters in the CG on day 14.
  
  
  CONCLUSIONS
  Using this disc pre-treatment, i.e., the surgical removal and controlled trypsinization of the outer AF, NP bioactivity was better maintained for up to 14 days in an in vitro disc bioreactor culture.
authors.affiliation: Department of Orthopedic Surgery, Southwest Hospital, Third Military Medical University, Gao Tan Yan 29, Chongqing, 400038, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Orthopedic Surgery, Kunming General Hospital of Chengdu Command, Kunming, Yunnan, 650032, China.
authors.forename: Rongmao
authors.initials: R
authors.lastname: Shi
authors.affiliation: The 91245 Troops of the Chinese People's Liberation Army, Huludao, Liaoning, 125000, China.
authors.forename: Daosen
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Orthopedic Surgery, Southwest Hospital, Third Military Medical University, Gao Tan Yan 29, Chongqing, 400038, China.
authors.forename: Yibo
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Department of Orthopedic Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing, 400038, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Orthopedic Surgery, Southwest Hospital, Third Military Medical University, Gao Tan Yan 29, Chongqing, 400038, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Song
authors.affiliation: Department of Orthopedic Surgery, No. 181 hospital of PLA, Guilin, Guangxi, 541002, China.
authors.forename: Songtao
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Orthopedic Surgery, Southwest Hospital, Third Military Medical University, Gao Tan Yan 29, Chongqing, 400038, China. zq_tlh@163.comAQ2.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D000818:Animals; D019149:Bioreactors; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D007403:Intervertebral Disc / Q000201:enzymology* / Q000473:pathology / Q000601:surgery; D008297:Male; D008751:Methylene Blue / Q000378:metabolism; D009924:Organ Culture Techniques / Q000295:instrumentation*; D011817:Rabbits; D013997:Time Factors; D020858:Tissue and Organ Harvesting; D014357:Trypsin / Q000378:metabolism*
chemical_list: D014357:Trypsin; D008751:Methylene Blue
keywords: Degeneration; Intervertebral disc; Nucleus pulposus; Organ culture