Speaker
Description
"Introduction: Endochondral ossification (EO) is the process of bone development via a cartilage template. It involves multiple stages, including chondrogenesis, mineralization and angiogenesis. Importantly, how angiogenesis contributes to EO is not fully understood. To characterise the interaction between human mesenchymal stromal cells(hMSCs)-derived cartilage and blood vessels, we designed an in vitro co-culture model comprised of tissue engineered hMSCs derived cartilage pellets that undergo mineralisation, and adipose-derived stromal cells (ASCs) and human umbilical vein endothelial cells (HUVECs) that form blood vessel structures. After characterisation of the pellets, we assessed the effect of their conditioned medium on angiogenesis using HUVECs migration and proliferation assays. Finally, we co-cultured the pellets, HUVECs and ASCs in a fibrin hydrogel to develop a comprehensive system to study cartilage vascularisation.
Methodology: Chondrogenic hMSC pellets (N = 3 donors) were generated by culture with medium containing transforming growth factor(TGF)-β3 (10ng/ml) for 28 days. For mineralized pellets, β-Glycerophosphate (BGP) (10mM) was added from day 7 and TGF-β3 was withdrawn on day 14. On days 7, 14, 21 and 28, conditioned media were produced by culturing the pellets for 24h in basal medium. Then, the pellets were harvested for gene expression and histological analysis. Thionine and Von Kossa stainings were performed to detect glycosaminoglycans (GAGs) and calcium deposits, respectively. Transwell migration and EdU-proliferation assays were employed to evaluate the effect of conditioned medium on HUVECs. To generate a 3D-vascular network, HUVECs and ASCs were simultaneously co-cultured with pellets in a fibrin hydrogel for 14 days. The vessel structures were visualised by immunofluorescent staining for laminin.
Results: Thionine and von Kossa staining evidenced successful in vitro cartilage formation and mineralisation, respectively. BGP exposure induced the formation of mineralised deposits which increased in time, while GAG staining progressively decreased. The mRNA expression of osteogenic (ALPL, IBSP) and angiogenic/remodelling markers (VEGFA, MMP13) in mineralised pellets showed the highest levels on d14 and decreased during late mineralisation. Transwell migration assays showed that conditioned medium from chondrogenic and mineralised pellets stimulates HUVEC migration (24.2-folds and 16.8-folds vs. negative control, respectively). HUVEC proliferation was also increased after exposure to conditioned medium from chondrogenic or mineralised pellets (1.9-fold and 2-fold vs. negative control, respectively). Finally, by co-culturing pellets/ASCs/HUVECs in a fibrin hydrogel, we achieved the successful formation of a 3D vascular network. Confocal imaging analyses revealed contact between microvessels and chondrogenic/mineralised pellets.
Conclusions: In this study, we established an in vitro model of cartilage vascularisation during endochondral ossification. By characterising mineralising pellet cultures, we found that the expression of pro-angiogenic markers and the pro-migratory and pro-proliferative effects towards HUVECs are maximum during early mineralisation and then decrease. Furthermore, 3D in vitro vascular network formation was achieved in the presence of chondrogenic or mineralised pellets. Our in vitro 3D co-culture model can be applied for mechanistic studies on the role of angiogenesis in bone formation and repair, as well as disease modelling."
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