title: A Nucleotomy Model with Intact Annulus Fibrosus to Test Intervertebral Disc Regeneration Strategies.
abstract: BACKGROUND
  New cells/hydrogel-based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but does not allow the injection of a significant volume into intact IVD. The aim of this study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical nucleotomy (N), or a combining technique (E+N), as a model to study IVD regeneration strategies with intact annulus fibrosus (AF) in functional spinal units (FSUs) in vitro.
  
  
  METHODS
  The transpedicular approach via the endplate route (2 mm tunnel) was performed on ovine FSU (IVD and superior and inferior endplate) to access the NP. FSUs were treated by N (Arthroscopic shaver), E (Trypsin/Collagenase), or E+N. Treatments were evaluated macro- and microscopically. The degradation of proteoglycan (PG) around the cavity was assessed by gel electrophoresis. Cell viability was evaluated using the lactate dehydrogenase (LDH) assay. Cavity volume was quantified through computerized tomography after injection of agarose gel/contrast agent.
  
  
  RESULTS
  A cavity with intact AF was successfully created with all three methods. The N group showed high reproducibility, low PG degradation, and no endplate thinning. Histological analysis demonstrated NP matrix degradation in enzyme-treated groups, while the PG content was homogenous using mechanical discectomy. Cell viability was affected only in the E group. The cavity volume normalized to the total IVD volume was 5.2% ± 1.6% in E, 5.0% ± 1.4% in E+N, and 4.2% ± 0.1% in N.
  
  
  CONCLUSIONS
  Mechanical nucleotomy leads to a more reproducible and less destructive cavity in the NP. Enzymatic methods perform better in terms of cavity volume; however, the cells and PG of the surrounding tissue may be affected. The mechanical nucleotomy enables the creation of a cavity into the IVD while keeping the AF intact, allowing the injection of reproducible volumes of hydrogel and tissue engineering construct for preclinical tests.
authors.affiliation: 1 Department of Orthopedic and Trauma Surgery, Campus Bio-Medico University of Rome , Rome, Italy .|2 Cell Factory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico , Milan, Italy .
authors.forename: Gianluca
authors.initials: G
authors.lastname: Vadalà
authors.affiliation: 1 Department of Orthopedic and Trauma Surgery, Campus Bio-Medico University of Rome , Rome, Italy .
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Russo
authors.affiliation: 3 AO Research Institute Davos , Davos, Switzerland .|4 INSERM UMRS 791, LIOAD, Research Center for Osteoarticular and Dental Tissue Engineering , Nantes, France .
authors.forename: Girish
authors.initials: G
authors.lastname: Pattappa
authors.affiliation: 3 AO Research Institute Davos , Davos, Switzerland .
authors.forename: Marianna
authors.initials: M
authors.lastname: Peroglio
authors.affiliation: 3 AO Research Institute Davos , Davos, Switzerland .
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Stadelmann
authors.affiliation: 5 Shriners Hospital , Montreal, Canada .
authors.forename: Peter
authors.initials: P
authors.lastname: Roughley
authors.affiliation: 3 AO Research Institute Davos , Davos, Switzerland .
authors.forename: Sibylle
authors.initials: S
authors.lastname: Grad
authors.affiliation: 3 AO Research Institute Davos , Davos, Switzerland .
authors.forename: Mauro
authors.initials: M
authors.lastname: Alini
authors.affiliation: 1 Department of Orthopedic and Trauma Surgery, Campus Bio-Medico University of Rome , Rome, Italy .
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Denaro
mesh_terms: D000818:Animals; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D007403:Intervertebral Disc / Q000000981:diagnostic imaging / Q000473:pathology / Q000601:surgery*; D008954:Models, Biological; D011509:Proteoglycans / Q000378:metabolism; D012038:Regeneration; D012756:Sheep; D014057:Tomography, X-Ray Computed
chemical_list: D011509:Proteoglycans