While mesenchymal stromal cells (MSCs) are a very attractive cell source for cartilage regeneration, an inherent tendency to undergo hypertrophic maturation and endochondral ossification; as well as insufficient extracellular matrix production still prevent their clinical application in cell –based cartilage repair therapies. We recently demonstrated that intermittent treatment of MSC with parathyroid hormone-related protein (PTHrP) during in vitro chondrogenesis significantly enhanced extracellular matrix deposition and concomitantly reduced hypertrophy (1) opposite to constant PTHrP treatment, which strongly suppressed chondrogenesis via the cAMP/PKA pathway (2). Since signal timing seemed to be decisive for an anabolic versus catabolic outcome of the PTHrP treatment, we here aimed to investigate the role of PTHrP pulse frequency, pulse duration and total weekly exposure time in order to unlock the full potential of PTHrP pulse application to enhance and control MSC chondrogenesis.

Human bone marrow-derived MSC were subjected to in vitro chondrogenesis for six weeks. From day 7–42, cells were additionally exposed to 2.5 nM PTHrP(1–34) pulses or left untreated (control). Pulse frequency was increased from three times per week (3×6h/week) to daily, thereby maintaining either pulse duration (6h/d, total 42 h/week) or total weekly exposure time (2.6h/d, total 18 h/week).

A high frequency of PTHrP-treatment (daily) was important to significantly increase extracellular matrix deposition and strongly suppress ALP activity by 87 %; independent of the pulse duration. A long pulse duration was, however, critical for the suppression of the hypertrophic marker gene IHH, while MEF2C and IBSP were significantly suppressed by all tested pulse duration and frequency protocols. COL10A1, RUNX2 and MMP13 mRNA levels remained unaffected by intermittent PTHrP. A drop of Sox9 levels and a decreased proliferation rate after 6 hours of PTHrP exposure on day 14 indicated delayed chondroblast formation. Decreased IGFBP-2, -3 and -6 expression as well as decreased IGFBP-2 protein levels in culture supernatants suggested IGF-I-related mechanisms behind anabolic matrix stimulation by intermittent PTHrP.

The significant improvement of MSC chondrogenesis by the optimization of intermittent PTHrP application timing revealed the vast potential of PTHrP to suppress hypertrophy and stimulate chondrogenic matrix deposition. A treatment with PTHrP for 6 hours daily emerged as the most effective treatment mode. IGF-I and Sox-9 related mechanisms are suggested behind anabolic effects and delayed chondroblasts formation, respectively. Thus, similar to the established osteoporosis treatment, daily injections of PTHrP may become clinically relevant to support cartilage repair strategies relying on MSCs like subchondral bone microfracturing and autologous MSC implantation.