gms | German Medical Science

Objectives: Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix and the avoidance of pre-mature hypertrophy being the prime challenges. As one of the master regulators of chondrocyte differentiation and hypertrophy, parathyroid hormone-related protein (PTHrP) is a promising candidate factor to direct the chondrogenesis of MSCs towards an articular phenotype. We recently demonstrated that while constant exposure to PTHrP suppressed MSC chondrogenesis, intermittent PTHrP application increased extracellular matrix production and concomitantly reduced undesired hypertrophy. Mechanistic analyses revealed PTHrP signal timing as critical variable for beneficial effects, thus, we here investigated the role of frequency, pulse duration and total exposure time of intermittent PTHrP in order to unlock its full potential.

Methods: Human bone marrow-derived MSCs were subjected to in vitro chondrogenesis for six weeks and were intermittently exposed to 2.5 nM PTHrP(1-34) from day 7-42. Application frequency of PTHrP was increased from three times weekly (3x6h/week, total exposure time 18h/week) to daily, thereby either maintaining the pulse duration (6h/day) or total weekly exposure time (18h/week, 2.6h/day).

Results and Conclusion: Enhanced frequency of PTHrP treatment significantly increased extracellular matrix deposition at the same time suppressing alkaline phosphatase (ALP) activity by 87%, regardless of pulse duration. Pulse duration, however, was critically important for a significant reduction of the hypertrophic marker gene IHH but irrelevant for suppression of MEF2C and IBSP. COL10A1, RUNX2 and MMP13 expression levels remained unaffected. A drop of Sox9 levels and a decreased proliferation rate after 6 hours of PTHrP exposure on day 14 correlated with delayed chondroblast formation and hypertrophy. Decreased IGFBP-2 expression in all groups suggested IGF-I-related mechanisms behind intermittent PTHrP anabolic actions.

Overall, the significant improvement of MSC chondrogenesis by the optimization of intermittent PTHrP application timing demonstrated the vast potential of PTHrP to suppress hypertrophy and stimulate chondrogenic matrix deposition. In view of this, daily injections of PTHrP, as already used for osteoporosis treatment, may become clinically relevant as supportive treatment for cartilage repair strategies, especially for those which rely on MSCs like microfracture and MSC implantation.