Endochondral ossification (EO) is the process of bone development via a cartilage template. It involves multiple stages, including chondrogenesis, mineralisation and angiogenesis. Importantly, how cartilage mineralisation affects angiogenesis during EO is not fully understood. Here we aimed to develop a new in vitro co-culture model to recapitulate and study the interaction between mineralised cartilage generated from human mesenchymal stromal cells (hMSCs) and microvascular networks. Chondrogenic hMSC pellets were generated by culture with transforming growth factor (TGF)-β3. For mineralised pellets, β-glycerophosphate (BGP) was added from day 7 and TGF-β3 was withdrawn on day 14. Conditioned medium (CM) from the pellets was used to evaluate the effect on human umbilical vein endothelial cells (HUVECs) in migration, proliferation and tube formation assays. To perform direct co-cultures, pellets were embedded in fibrin hydrogels containing vessel-forming cells (HUVECs, adipose stromal cells) for 10 days with BGP to induce mineralisation. The pellets and hydrogels were characterised by immunohistochemistry and confocal imaging.Introduction
Method
Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2); Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3); Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes.
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