Phenotype instability and premature hypertrophy prevent the use of human mesenchymal stromal cells (MSCs) for cartilage regeneration. Aim of this study was to investigate whether intermittent supplementation of parathyroid hormone-related protein (PTHrP), as opposed to constant treatment, can beneficially influence MSC chondrogenesis and to explore molecular mechanisms below catabolic and anabolic responses. Human MSCs subjected to chondrogenic induction in high-density culture received PTHrP(1-34), forskolin, dbcAMP, or PTHrP(7-34) either constantly or via 6-h pulses (three times weekly), before proteoglycan, collagen type II, and X deposition; gene expression; and alkaline phosphatase (ALP) activity were assessed. While constant application of PTHrP(1-34) suppressed chondrogenesis of MSCs, pulsed application significantly increased collagen type 2 (COL2A1) gene expression and the collagen type II, proteoglycan, and DNA content of pellets after 6 weeks. Collagen type 10 (COL10A1) gene expression was little affected but Indian hedgehog (IHH) expression and ALP activity were significantly downregulated by pulsed PTHrP. A faster response to PTHrP exposure was recorded for ALP activity over COL2A1 regulation, suggesting that signal duration is critical for catabolic versus anabolic reactions. Stimulation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling by forskolin reproduced major effects of both treatment modes, whereas application of PTHrP(7-34) capable of protein kinase C (PKC) signaling was ineffective. Pulsed PTHrP exposure of MSCs 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 MSCs and may be considered as a supporting clinical-treatment mode for MSCbased cartilage defect regeneration.

Human mesenchymal stromal cells (hMSC) differentiating toward the chondrogenic lineage recapitulate successive phases of embryonic chondrocyte maturation developing from progenitor cells to hypertrophic chondrocytes. Osteoarthritic cartilage is characterized by an alteration in chondrocyte metabolism and upregulation of hypertrophic differentiation markers. A number of studies point toward a functional role for microRNAs (miRs) in controlling chondrocyte differentiation and development of osteoarthritis (OA). However, information on miRs that may regulate a specific phase of chondrocyte maturation, especially hypertrophy, is lacking. We here aimed to unravel miR profiles modulated during chondrogenesis of hMSC to obtain new differentiation markers and potential new targets relevant for differentiation outcome and OA development. hMSC were subjected to transforming growth factor-β (TGF-β)-driven chondrogenesis and miR profiles were determined by microarray analysis at distinct developmental time points. Expression of selected miRs was compared to cultures lacking chondrogenesis and to redifferentiated nonhypertrophic articular chondrocytes. Among 1349 probed miRs, 553 were expressed and 169 (31%) were significantly regulated during chondrogenesis. Hierarchical clustering identified specific miR expression patterns representative for MSC, prechondrocytes, chondroblasts, chondrocytes, and hypertrophic chondrocytes, respectively. Regulation of miR-181 family members allowed discrimination of successive differentiation stages. Levels of several miRs, including miR-23b, miR-140, miR-181, and miR-210 positively correlated with successful chondrocyte formation. Hypertrophic MSC-derived chondrocytes and nonhypertrophic articular chondrocytes showed differential expression of miR-181a, miR-210, and miR-31, but not miR-148a implicated in COL10A1-regulation. We conclude that the here identified stage-dependent miR clusters may have imperative functions during chondrocyte differentiation providing novel diagnostic tools and targets of potential relevance for OA development.

Aim of this study was a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode and characterization of time evolution and re-inducibility of the response. Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to β-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9 × 10 min over 3 hr) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35 S-sulfate-incorporation over 24 hr. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p = 0.016). Among 115 significantly regulated genes, 114 were upregulated, 48 of them ≥ twofold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression.Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-β-, calcium-, retinoic-acid-, Wnt-, and Notchsignaling pathway which were significantly upregulated SOX9, and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signaling showed no consistent regulation. This study associates novel genes with mechanoregulation in chondrocytes, raising SOX9 protein levels with anabolic loading and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Upregulation of mechanosensitive indicators extending differentially into the resting time provides crucial knowledge to maximize cartilage matrix deposition for the generation of high-level cartilage replacement tissue.