title: Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells.
abstract: BACKGROUND
  The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection.
  
  
  OBJECTIVE
  To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo.
  
  
  METHODS
  In vitro and ex vivo whole-organ culture of hMSCs.
  
  
  METHODS
  In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype.
  
  
  RESULTS
  The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs.
  
  
  CONCLUSIONS
  Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.
authors.affiliation: AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland. Electronic address: marianna.peroglio@aofoundation.org.
authors.forename: Marianna
authors.initials: M
authors.lastname: Peroglio
authors.forename: David
authors.initials: D
authors.lastname: Eglin
authors.forename: Lorin M
authors.initials: LM
authors.lastname: Benneker
authors.forename: Mauro
authors.initials: M
authors.lastname: Alini
authors.forename: Sibylle
authors.initials: S
authors.lastname: Grad
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D020100:Hydrogels / Q000494:pharmacology*; D007403:Intervertebral Disc; D059630:Mesenchymal Stem Cells / Q000187:drug effects*
chemical_list: D020100:Hydrogels; D006820:Hyaluronic Acid
keywords: Human mesenchymal stem cells; Hyaluronan; Intervertebral disc; Thermoreversible hydrogel; Whole-organ culture