title: Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin-hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution.
abstract: OBJECTIVE
  Cell therapy would be favorably performed immediately after nucleotomy, to restore intervertebral disc functionality and to slow down disc degeneration. Promising results were reported from small animal models but remaining problems, especially in larger animals, include loss of vital cells due to annular damage at the injection site and detrimental intradiscal conditions. The aim of the present study was to optimize cell-based disc therapy using a new albumin-hyaluronan hydrogel together with bone marrow-derived mesenchymal stem cells in a large porcine disc model.
  
  
  METHODS
  Luciferase cell labeling was evaluated to follow-up stem cells metabolically up to 7 days in 3D cell cultures mimicking the harsh disc environment with low oxygen and glucose concentrations. As a pilot in vivo study, the implant was injected into porcine discs after removal of ~10% of nucleus volume and animals were killed immediately after surgery (n = 6) and 3 days later (n = 6). 24 discs were analyzed. Implant persistence and cell activity (luciferase + WST assay) were observed simultaneously.
  
  
  RESULTS
  In vitro cell culture with reduction of glucose (20, 5, 0.5, 0 mM) and oxygen (21, 5, 2%) significantly decreased metabolic cell activity and luciferase activity after 3 days, with no recovery and a further decrease after 7 days, establishing luciferase activity as a metabolic sensor. During 3 days of 3D culture with disc-like conditions, luciferase activity decreased to 8%. In vivo, initial implant volume shrank to 61% at day 3 with evidence for hydrogel compression. Luciferase activity in vivo at day 3 was 2% without referencing but 23% after referencing to in vitro cell adaptation, and 38% after additional consideration of detected implant volume loss.
  
  
  CONCLUSIONS
  In vitro analysis up to 7 days established for the first time luciferase activity as a metabolic sensor for mesenchymal stem cells used in regenerative disc therapy. Under the present protocol, short-term in vivo analysis after 3 days suggests improved implant retainment inside the disc and persistence of metabolically active cells; however, further studies will have to prove long-term in vivo outcome.
authors.affiliation: Clinic for Orthopaedics and Trauma Surgery, Heidelberg University Hospital, Schlierbacher Landstr. 200a, 69118, Heidelberg, Germany, georgomlor@gmx.de.
authors.forename: G W
authors.initials: GW
authors.lastname: Omlor
authors.forename: J
authors.initials: J
authors.lastname: Fischer
authors.forename: K
authors.initials: K
authors.lastname: Kleinschmitt
authors.forename: K
authors.initials: K
authors.lastname: Benz
authors.forename: J
authors.initials: J
authors.lastname: Holschbach
authors.forename: K
authors.initials: K
authors.lastname: Brohm
authors.forename: M
authors.initials: M
authors.lastname: Anton
authors.forename: T
authors.initials: T
authors.lastname: Guehring
authors.forename: W
authors.initials: W
authors.lastname: Richter
mesh_terms: D000418:Albumins / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques; D004195:Disease Models, Animal; D017586:Diskectomy / Q000379:methods*; D005500:Follow-Up Studies; D005947:Glucose / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000494:pharmacology*; D007403:Intervertebral Disc / Q000000981:diagnostic imaging / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000000981:diagnostic imaging / Q000601:surgery / Q000628:therapy; D008156:Luciferases; D008159:Lumbar Vertebrae; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology; D010100:Oxygen / Q000378:metabolism; D013552:Swine; D014057:Tomography, X-Ray Computed
chemical_list: D000418:Albumins; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D006820:Hyaluronic Acid; D008156:Luciferases; D005947:Glucose; D010100:Oxygen