Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs

Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material 3D bioprinting can potentially resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge. Here, we developed endothelial cell(EC)-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM). EC-driven capillary network formation started two days after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29% after 14 days, compared to col-1 MFs-laden bioinks. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a microgel suspension bath. The constructs were cultured up to 14 days, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models, cancer progression, and testing anti-angiogenic therapies.