The efficiency of engineered bone grafts (mesenchymal stromal cells MSCs and biomaterial) in aiding bone repair in non-union fractures is impeded by inadequate vascularization. MSCs and endothelial cells (ECs) co-cultures have been investigated to promote rapid vascularization. However, co-cultures further complicate the process. Therefore, the development of vascularized bone grafts deriving from a single tissue source is necessary.

The patented procedure (FR2102708), developed by the French Blood Establishment-Flames Team (Toulouse), demonstrated the concurrent selection and amplification of MSCs and ECs from bone marrow mononuclear cells (BMMCs) of femoral head with EGM2. These cells were then subjected to a 3D multi-differentiation spheroidal culture, resulting in a human bone marrow model presenting adipocytic, osteoblastic compartments and vascularization networks. In an effort to render the amplification medium more suitable for clinics, the current studies consist of further optimizing the amplification medium to increase the proliferation rate and maximize the vasculature level by replacing the fetal bovine serum (FBS) in EGM2 with PL (EGM2+PL).

To reach our objective, BMMCs were cultured in EGM2+PL. The resulting cell populations were then subjected to a 14-day 3D multi-differentiation spheroidal culture.

First, we showed that EGM2+PL enhance cell proliferation in comparison to the EGM2 and the current clinical amplification medium (αMEM+PL). RT-qPCR results indicate some expressions of endothelial markers at T0, suggesting EGM2+PL can maintain EPCs population during the amplification phase. Next, the cellular organization within the spheroid is determined by immunofluorescence at T3, T7 and T14 of the multi-differentiation spheroidal culture. Vessel-like networks were observed as early as T3 and the network matures over time. Moreover, nestin positive cells are observed closely bound to the vessels, highlighting the presence of pericytes supporting ECs network. From recent preliminary results of in vivo experiments, we observed the formation of mature bone following 8-week subcutaneous graft of spheroids with biomaterials.

To conclude, we have generated vascularized bone spheroids from a single primary tissue source. To go further, the functionality of these spheroids in vivo should be further investigated.