Melatonin contributes to the hypertrophic differentiation of mesenchymal stem cell-derived chondrocytes via activation of the Wnt/β-catenin signaling pathway

Background Hypertrophy is a critical process for chondrocyte differentiation and maturation during endochondral ossification, which is responsible for the formation of long bone and postnatal longitudinal growth. Increasing evidence suggests that melatonin, an indole hormone, plays a pivotal role in chondrogenesis. However, little is known about the effects of melatonin on the terminal differentiation of chondrocytes. Methods Mesenchymal stem cell (MSC)-derived chondrocytes generated by a high-density micromass culture system were induced to undergo hypertrophic differentiation. Melatonin-mediated hypertrophic differentiation was examined by reverse transcription polymerase chain reaction analysis (RT-PCR) analysis, histological staining and immunohistochemistry. Activation of the Wnt signaling pathway was evaluated by PCR array, RT-PCR, western blotting and immunofluorescence. XAV-939, a Wnt signaling pathway antagonist, was further used to determine whether the effect of melatonin on chondrocyte hypertrophic differentiation was mediated occurred by activation of Wnt signaling pathway. Results Histological staining showed melatonin increased chondrocyte cell volume and the expression of type X collagen but decreased the expression of type II collagen compared with the control group. RT-PCR showed that melatonin significantly up-regulated the gene expressions of biomarkers of hypertrophic chondrocytes, including type X collagen, alkaline phosphatase, runt-related transcription factor 2, Indian hedgehog and parathyroid hormone-related protein receptor, and melatonin down-regulated the mRNA expression of hallmarks of chondrocytes, including parathyroid hormone-related protein. PCR array showed that the effect of melatonin on chondrocyte hypertrophic differentiation was accompanied by the up-regulation of multiple target genes of the canonical Wnt signaling pathway, and this effect was blocked by XAV-939. Conclusions The current findings demonstrate that melatonin enhances the hypertrophic differentiation of MSC-derived chondrocytes through the Wnt signaling pathway. Our findings add evidence to the role of melatonin in promoting bone development and highlight the positive effects of melatonin on terminal differentiation of chondrocytes.

Melatonin Contributes to The Hypertrophic Differentiation of Mesenchymal Stem Cell-Derived Chondrocytes Via Activation of The WNT/β-Catenin Signaling Pathway

BackgroundHypertrophy is a critical process for chondrocyte differentiation and maturation during endochondral ossification, which is responsible for the formation of long bone and its postnatal longitudinal growth. Increasing evidence suggests that melatonin, an indole hormone, plays a pivotal role in chondrogenesis; however, little is known about its effects on the terminal differentiation of chondrocytes. MethodsMesenchymal stem cells (MSCs) derived chondrocytes generated by a high-density micromass culture system were further induced to hypertrophic differentiation. The melatonin-mediated hypertrophic differentiation were examined by reverse transcription polymerase chain reaction analysis (RT-PCR) analysis and histological staining and immunohistochemistry. The expression of downstream factors of WNT signaling pathway was evaluated by RT-PCR, western blotting and immunofluorescence. The WNT signaling pathway antagonist XAV-939 was used to further demonstrate melatonin-induced hypertrophic chondrocytes and WNT signaling pathway activation.ResultsHistological staining showed melatonin increased the chondrocytes cell volume and the expression of type X collagen, but decreased the expression of type II collagen compare to the control group. RT-PCR showed that melatonin significantly up-regulated the expression of biomarkers of hypertrophic chondrocytes, including type X collagen (COL10A1), alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), Indian hedgehog (IHH), and parathyroid hormone-related protein receptor (PTHrP-R) and down-regulated the hallmarks of chondrocytes, including parathyroid hormone-related protein (PTHrP). The WNT signaling pathway PCR array showed that the effect of melatonin was accompanied by the up-regulation of multiple target genes of the canonical WNT signaling pathway and the melatonin-mediated effect can be blocked by XAV-939. ConclusionsThese findings demonstrate that melatonin can enhance the hypertrophic differentiation of MSCs-derived chondrocytes through the WNT signaling pathway. It adds evidence to the role of melatonin in promoting bone development and highlights its positive effects on chondrocytes terminal differentiation.

Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion

ObjectiveHuman nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites.MethodsHuman NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsCoculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.