gms | German Medical Science

Objectives: The use of human mesenchymal stromal cells (MSC) for cartilage regeneration is prevented by phenotype instability and pre-mature hypertrophy. Aim of this study was to investigate whether intermittent application of parathyroid hormone-related protein (PTHrP) can, in contrast to constant treatment, positively influence MSC chondrogenesis and to explore the molecular mechanisms behind differential responses to both treatment modes.

Methods: Human MSC were subjected to chondrogenic induction in high-density pellet culture for 6 weeks. Cells received either chondrogenic medium alone (control) or supplemented with PTHrP(1-34), forskolin, dbcAMP or PTHrP(7-34) either constantly or via 6 hour pulses (3-times weekly). Proteoglycan and DNA content, collagen type II and -X deposition, gene expression of chondrogenic and hypertrophic markers and alkaline phosphatase (ALP) activity were quantitatively assessed at different time points during chondrogenic induction.

Results and Conclusion: Constant application of PTHrP(1-34) suppressed chondrogenesis of MSC, whereas pulsed application significantly increased collagen type II gene (COL2A1) expression and collagen type II deposition, as well as proteoglycan and DNA content of pellets after 6 weeks. Collagen type X gene and protein expression levels were not altered, but indian hedgehog (IHH) gene expression and ALP activity were significantly reduced by pulsed PTHrP. Stimulation of cAMP/PKA signaling by forskolin reproduced major effects of both treatment modes, whereas application of the N-terminally truncated PTHrP(7-34), capable to activate PKC- but not cAMP/PKA signalling, was ineffective.

Pulsed PTHrP exposure of MSC stimulated chondrogenesis and reduced endochondral differentiation apparently uncoupling chondrogenic matrix deposition from hypertrophic marker expression. cAMP/PKA was the major signaling-pathway triggering the opposing effects of both treatment modes. Intermittent application of PTHrP represents an important novel means to improve chondrogenesis of MSC and may be considered as a supporting clinical treatment mode for MSC-based cartilage defect regeneration.