title: An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue.
abstract: BACKGROUND
  The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.
  
  
  METHODS
  Human MSCs extracted from bone marrow were expanded in monolayer culture and, after labelling with adenoviral vectors carrying the green fluorescent protein transcript, were injected into NP tissue explants (sourced from bovine caudal discs) and maintained in culture for 2, 7, 14 and 28 days post injection. Following fixation and paraffin embedding, cell viability was assessed using in situ hybridisation for polyA-mRNA and using immunohistochemistry for caspase 3. Immunohistochemistry/fluorescence for aggrecan, Sox-9 and types I, II and X collagen together with Alizarin red staining was employed to investigate the MSC phenotype and matrix formation.
  
  
  RESULTS
  MSCs were identified in all injected tissue samples and cell viability was maintained for the 4 weeks investigated. MSCs displayed cellular staining for Sox-9, and displayed cellular and matrix staining for aggrecan and type II collagen that increased during culture. No type I collagen, type X collagen or Alizarin red staining was observed at any time point.
  
  
  CONCLUSIONS
  MSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in vitro, and thus may not require additional stimulation or carrier to induce differentiation. This is a key finding, as such a strategy would minimise the level of external manipulation required prior to insertion into the patient, thus simplifying the treatment strategy and reducing costs.
authors.affiliation: Biomedical Research Centre, Biosciences, Faculty of Health and Wellbeing, Sheffield Hallam University, City Campus, Owen Building, Howard Street, Sheffield S11WB, UK. c.lemaitre@shu.ac.uk
authors.forename: Christine L
authors.initials: CL
authors.lastname: Le Maitre
authors.forename: Pauline
authors.initials: P
authors.lastname: Baird
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Freemont
authors.forename: Judith A
authors.initials: JA
authors.lastname: Hoyland
mesh_terms: D000368:Aged; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival; D005109:Extracellular Matrix / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D007403:Intervertebral Disc / Q000166:cytology*; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010641:Phenotype