Aims

Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism.

Objectives

Previously, we reported the improved transfection efficiency of a plasmid DNA-chitosan (pDNA-CS) complex using a phosphorylatable nuclear localization signal-linked nucleic kinase substrate short peptide (pNNS) conjugated to chitosan (pNNS-CS). This study investigated the effects of pNNS-CS-mediated miR-140 and interleukin-1 receptor antagonist protein (IL-1Ra) gene transfection both in rabbit chondrocytes and a cartilage defect model.

Adult chondrocytes experience a hypoxic environment in vivo. Culturing chondrocytes under oxygen tension that more closely resembles the in vivo situation, i.e. hypoxic conditions, has been shown to have positive effects on matrix synthesis. During redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances redifferentiation is still incompletely elucidated. We employed micro-bioreactor technology to elucidate the contribution of TGF-β superfamily ligands to the chondrocyte differentiation process under hypoxic conditions in vitro. Dedifferentiated chondrocytes in alginate were cultured for 48 hours under hypoxic (1% pO2) or normoxic (20%) conditions, using specialized bioreactor technology. Gene expression of chondrocyte-specific markers (SOX9, COL2A1, COL1A1, AGC1 and MMP13) as well as established hypoxia-controlled genes (GDF1-, PHD3, HAS2, VEGF, COX2) and components of the TGF-β superfamily signaling pathways were analyzed by qPCR and protein expression after 48 hours in combination with TGF-β superfamily ligand-specific siRNA as well as selected TGF-β superfamily receptor inhibitors. Hypoxic culture showed robust upregulation of the selected hypoxia-specific marker genes. In addition, well-established chondrocyte-specific markers like SOX9 and collagen type II were upregulated. TGF-β isoforms were selectively upregulated under hypoxia on both mRNA and protein level. In addition, both Activin receptor-like kinases, ALK1 and ALK5, were upregulated under hypoxia, while respective type II and III receptors were unresponsive. The hypoxia-induced COL2 expression was abrogated by TGF-β2 siRNA, as was ALK5 inhibition. Our data strongly indicates that TGF-β superfamily signaling pathways are involved in chondrocyte redifferentiation under low oxygen tension in vitro

Mesenchymal stromal cells (MSC) are multipotent, self-renewing cells that are an attractive cell source for cartilage regeneration strategies. While articular chondrocytes form stable cartilage-like tissue under chondrogenic in vitro conditions, a still unsolved problem of chondrocyte production from MSC is their endochondrol development leading to the formation of transient instead of stable articular cartilage. In order to identify relevant molecular determinants of chondrocyte redifferentiation versus MSC chondrogenesis and hypertrophy, this study assessed the differential expression of members of the transforming growth factor β (TGF-β) -superfamily, their receptors and antagonists between differentiating MSC and human articular chondrocytes (HAC). Chondrogenesis of human MSC and redifferentiation of HAC was induced in micromass pellet culture. Gene expression of MSC (n=5) and HAC (n=5) was compared using a transcriptome analysis on Illumina platform. Functional regulation of relevant candidate molecules was assessed in independent MSC and HAC populations by qRT-PCR. Smad signalling during chondrogenic differentiation was analysed by immunohistochemistry and Western Blotting. BMP signalling in both populations was modulated by co-treatment with BMP-4/7 or an inhibitor of Smad1/5/9 signalling. Proteoglycan and DNA content, collagen type II and -X deposition, gene expression of chondrogenic and hypertrophic markers as well as alkaline phosphatase (ALP) activity were quantitatively assessed at different time points. In HAC, TGF-β receptor 2 and 3 (TGFBR2/3) were up-regulated to significantly higher levels than in MSC. BMP4, expressed during HAC expansion, was suppressed while CHL2 and CHRD levels raised. In MSC, BMP4 and BMP7 were induced while TGFBR2 and TGFBR3 were down-regulated. Staining for pSmad1/5/9 in HAC demonstrated positive cells dispersed throughout the pellets at day 3 and 5 while lower pSmad1/5/9 immunostaining was observed in MSC. In HAC and MSC pellets pSmad staining decreased during chondrogenesis, in line with Western Blot results. Medium supplementation with BMP-4/7 did not improve cartilaginous matrix deposition by MSC but raised ALP-activity. When Smad1/5/9 phosphorylation was blocked in MSC culture by dorsomorphin treatment (day 14–42) COL2A1 and COL10A1 expression decreased significantly and collagen type II and type X deposition were reduced. ALP activity dropped to 12 % of control levels. Inhibition of pSmad1/5/9 signalling was unattractive to shift chondrogenesis of MSC away from endochondral development since it unpaired SOX9 expression and strongly reduced cartilaginous matrix deposition along with hypertrophy. Thus no simple correlation exists between beneficial pSmad2/3 versus unwanted pSmad1/5/9 signalling during MSC chondrogenesis