Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential via modulation of culture conditions and Wnt/β-catenin pathway

Abstract

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate articular cartilage, but current chondroinduction protocols, commonly using transforming growth factor-β (TGFβ), lead to concomitant chondrocytic hypertrophy with ossification risk. Here, we showed that a 14-day culture of MSCs-laden hyaluronic acid hydrogel in the presence of TGFβ, followed by 7 days culture in TGFβ-free medium, with the supplement of Wnt/β-catenin inhibitor XAV939 from day 10–21, resulted in significantly reduced hypertrophy phenotype. The stability of the hyaline phenotype of the MSC-derived cartilage, generated with a standard protocol (Control) or the optimized (Optimized) method developed in this study, was further examined through intramuscular implantation in nude mice. After 4 weeks, constructs from Control group showed obvious mineralization; in contrast, the Optimized group displayed no signs of mineralization, and maintained cartilaginous histology. Further analysis showed that TGFβ treatment time affected p38 expression, while exposure to XAV939 significantly inhibited P-Smad 1/5 level, which together resulted in decreased level of Runx2. These findings suggest a novel treatment regimen to generate hyaline cartilage from human MSCs-loaded scaffolds, which have a minimal risk of eliciting endochondral ossification.

Significance of MEF2C and RUNX3 Regulation for Endochondral Differentiation of Human Mesenchymal Progenitor Cells

“…Detection of high RUNX2 protein levels at start of chondrogenesis (Deng et al, 2018) is in apparent contradiction to our work. However, Deng et al (2018) started MPC cultures, not from bone marrow aspirates but sieved cells from curetted and minced trabecular bone. By this, their cells expressed very strong RUNX2 protein levels after expansion, indicating a high osteoblast contamination of the used "MPC" population, which furthermore did not upregulate RUNX2 during chondrogenesis (Deng et al, 2018).…”

“…So far, only one group has visualized RUNX2 protein in human MPC chondrogenesis in photopolymerized hydrogels by Western blotting and suggested its downregulation under WNT pathway inhibition (Deng et al, 2018). Detection of high RUNX2 protein levels at start of chondrogenesis (Deng et al, 2018) is in apparent contradiction to our work. However, Deng et al (2018) started MPC cultures, not from bone marrow aspirates but sieved cells from curetted and minced trabecular bone.…”

“…Thus, Runx2 appeared more important for hypertrophy of cartilage than Runx3 in mice (Yoshida et al, 2004). This may be the reason why, to date, all publications on human MPC chondrogenesis addressing RUNT transcription factors reported only on RUNX2 as a prohypertrophic factor, while RUNX3 has remained so far unaddressed (Mueller and Tuan, 2008;Cooke et al, 2011;Caron et al, 2013;Karl et al, 2014;Correa et al, 2015;Dexheimer et al, 2016;Fan et al, 2016;Fischer et al, 2016;De Kroon et al, 2017;Deng et al, 2018;Diederichs et al, 2019;Kim et al, 2019). Most of these studies analyzed RUNX2 gene expression on a single (Cooke et al, 2011;Correa et al, 2015;Dexheimer et al, 2016;Fan et al, 2016;Fischer et al, 2016) or multiple timepoints during MPC chondrogenesis (Mueller and Tuan, 2008;Caron et al, 2013;Karl et al, 2014;De Kroon et al, 2017;Diederichs et al, 2019).…”

Click functionalized, tissue‐specific hydrogels for osteochondral tissue engineering

“…Additionally, all conditions except for BMHP1/PdBT (High) and GHK/PdBT (High) showed an increase in chondrogenic SOX9 expression at day 35. The lack of hypertrophic calcification within CS/PdBT and NC/PdBT hydrogels is beneficial given that many conventional methods of chondrogenic induction, such as transforming growth factor-β supplementation, produce unwanted conversion of hypertrophic chondrocytes to osteoblasts(Deng et al, 2019). Acellular hydrogels that were cultured under the same conditions F I G U R E 4 Histological imaging of 10 μm sections from MSC-laden hydrogels cultured for 35 days, showing cell content (red/purple), sGAG deposition (blue), and mineralization (brown/black).…”

“…methods of chondrogenic induction, such as transforming growth factor-β supplementation, produce unwanted conversion of hypertrophic chondrocytes to osteoblasts(Deng et al, 2019). Furthermore, both CS/PdBT and NC/PdBT inhibited gene expression associated with the osteogenic phenotype, while NC/PdBT (Low) in particular exhibited time-dependent upregulation of the early and mid/late stage chondrogenic markers.…”