title: The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions.
abstract: Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules--dexamethasone, triiodothyronine (T3) and insulin--on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.
authors.affiliation: Biomedical Laboratories, Swiss Paraplegic Center, G.A. Zaech Strasse 4, Nottwil, Switzerland.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bertolo
authors.forename: Ladina
authors.initials: L
authors.lastname: Ettinger
authors.forename: Niklaus
authors.initials: N
authors.lastname: Aebli
authors.forename: Daniel
authors.initials: D
authors.lastname: Haschtmann
authors.forename: Martin
authors.initials: M
authors.lastname: Baur
authors.forename: Ulrich
authors.initials: U
authors.lastname: Berlemann
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ferguson
authors.forename: Jivko V
authors.initials: JV
authors.lastname: Stoyanov
mesh_terms: D000328:Adult; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000464:Alginates; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003710:Demography; D003907:Dexamethasone / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000378:metabolism; D006603:Hexuronic Acids; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology*; D007403:Intervertebral Disc / Q000187:drug effects* / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D014284:Triiodothyronine / Q000494:pharmacology*
chemical_list: D053674:Aggrecans; D000464:Alginates; D024043:Collagen Type II; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D007328:Insulin; D014284:Triiodothyronine; D003907:Dexamethasone; D020723:Glucuronic Acid; D004247:DNA