title: Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.
abstract: BACKGROUND
  Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family.
  
  
  METHODS
  Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry.
  
  
  RESULTS
  Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs.
  
  
  CONCLUSIONS
  We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland. Samantha.chan@empa.ch.|Biointerfaces, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, St Gallen, CH-9014, Switzerland. Samantha.chan@empa.ch.
authors.forename: Samantha C W
authors.initials: SC
authors.lastname: Chan
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland. Adel.tekari@istb.unibe.ch.
authors.forename: Adel
authors.initials: A
authors.lastname: Tekari
authors.affiliation: Department for Orthopedic Surgery and Traumatology, Inselspital, University of Bern, Freiburgstrasse 4, Bern, CH-3010, Switzerland. lorin.benneker@insel.ch.|AOSpine Research Network, Stettbachstrasse 6, Dübendorf, CH-8600, Switzerland. lorin.benneker@insel.ch.
authors.forename: Lorin M
authors.initials: LM
authors.lastname: Benneker
authors.affiliation: Orthopedic Department, Sonnenhof Clinic, Buchserstrasse 30, Bern, CH-3006, Switzerland. Paul.Heini@sonnenhof.ch.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Heini
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland. Benjamin.Gantenbein@istb.unibe.ch.|AOSpine Research Network, Stettbachstrasse 6, Dübendorf, CH-8600, Switzerland. Benjamin.Gantenbein@istb.unibe.ch.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein
mesh_terms: D000328:Adult; D000369:Aged, 80 and over; D002113:Calcification, Physiologic / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000378:metabolism*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction
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